私立大学戦略的研究基盤形成支援事業（平成 23 年～27 年）
MEXT– supported Program for the Strategic Research
Foundation at Private Universities (2011－2015)

分子イメージングによる体系的病態の解明と診断治療法の開発
Systematic elucidation of pathogenesis anddevelopment of diagnosis and treatment bymolecular imaging
平成 23-24 年度研究成果中間報告書
Interim Report 2011-12

枚方学舎と病態分子イメージングセンター (MICD) からみた夜の枚方病院
24/7 isn't the only way: A healthy work–life balance can enhance research

関西医科大学
Kansai Medical University

平成 25 年 9 月
September 2013

“24/7 isn't the only way: A healthy work–life balance can enhance research”の意味するところは Nature 477: 20-22 & 27-28 (2011) を参照下さい。

目次Contents
はじめに
I．

Preface1

病態分子イメージングセンターの事業概要

3

Outline of Molecular Imaging Center of Diseases (MICD)
II．

研究活動の進捗状況・研究成果の要約

11

Activities and progress in research of MICD
III．

原著論文リスト

IV．

セミナー、シンポジウム、できごと

List of original articles

18
29

Seminars, symposia and events of MICD
 Program of the 1st annual meeting35

 Program of the 2nd annual meeting37

 Evaluation by advisory board members

38

資料

Supplementary Information

１．病態分子イメージングセンター組織運営規則

41

Organization and administrative regulation of MICD
２．第１回病態分子イメージングセンターAnnual meeting 抄録

43

Proceedings of the 1st annual meeting
３．第２回病態分子イメージングセンターAnnual meeting 抄録

53Proceedings of the 2nd annual meeting４．平成 23 年度研究成果報告

61

Research activities 2011５．平成 24 年度研究成果報告
Research activities 2012123

Figure 1. Identification of stem cells in lingual epithelium by multicolor lineage tracing
method. (A) Time course of lingual epithelial progenitor cells in the interpapillary pit (IPP) labeledwith multiple colors using rainbow mice. Rosa26CreERT2/rbw mice were treated with tamoxifen andanalyzed at the indicated time points. The IPP gained monoclonality at day 28.

(B)

Immunostaining of the tongue epithelium with Keratin 5 and Ki67 antibody and in situ hybridization,with a Bmi1 probe.

Schematic presentation of filliform papillae and magnified image of the IPP.

(C) Fate of cells derived from Bmi-positive stem cells. Bmi1CreER/+/Rosa26rbw/+ mice were injectedwith tamoxifen and analyzed at the indicated time points. (D) Cover of Nature Cell Biology 15 (5).
Bmi1-expressing cells ensure the maintenance and regeneration of keratinized cells in the murine
lingual epithelium.
Figure 2. Restricted expression of Ovol2/MOVO in XY body of mouse spermatocytes at thepachytene stage. (A) Schematic presentation of spermatogenesis. (B) Expression of Ovol2/MOVO
in the XY body. (Upper panel) Immunostaining of mouse testis with anti-Ovol2/MOVO andanti-H2AX antisera. (Lower panel) Ovol2/MOVO expression in seminiferous tubules at thedifferent stages of spermatogenesis. (C) Ovol2/MOVO expression of spermatocytes at pachytene
stage. Squash and spread spermatocytes were immunostained with anti-Ovol2/MOVO, anti-H2AX,
and anti-SYCP3 antibodies.
Figure 3. Analysis of thymocyte trafficking and antigen recognition by Mst1 in the thymus by
two-photon microscope.

(A) The experimental design for analysis of thymocyte migration insliced thymic tissues using two-photon microscope.

(B) Mst1+/+ or Mst1-/--labeled double positiveand single positive cells cultured on the thymic slice for several hours were observed within thethymic slice by a two-photon microscope.

(C) Antigen-specific thymocyte cluster formation in thethymic medulla of RIP-mOVA mice.
Figure 4. Visualization of acetylcholine distribution in the brain by imaging mass
spectrometry. (A) MS/MS spectra of m/z146 obtained from the ACh standard (left) and on brain
tissue (right). (B) a-c. Reconstructed ion intensity maps for the ion transition from m/z146 > 87 (a),in situ hybridization with ACh esterase (b), and scheme of the mouse brain map from the Atlas (c).
d-f. Expanded ACh distribution image of the hippocampus of an ISF-treated brain (d) and an opticalimage of the same brain section stained with H & E (e) and scheme of corresponding brain image(f).
Figure 5. Analysis of microfibril formation by latent TGF- binding protein 4 (LTBP-4). (A) A
proposed model for the role of LTBP-4-fibulin-5 interaction in elastic fiber assembly. (B)Recombinant LTBP-4S protein (rLTBP-4S) promotes elastic fiber assembly without changing theconfiguration of microfibrils.

Fig.1ABCD

図1. 多細胞系譜追跡法による舌毛状乳頭の幹細胞の同定 (Nat. Cell Biol. 15:511-518, 2013)

Fig.2
A

C

B

図2. 精巣特異的転写因子Ovol2の精子形成におけるパキテン期のXY体での発現 (J. Androl. 33:277-286, 2012)

Fig.3A

B

C

図3. 二光子励起顕微鏡を用いたMst1による胸腺細胞のトラフィッキングと抗原認識の解析(Nat. Commun. 3:1098, 2012)

Fig.4
A

B

図4. 質量顕微鏡を用いた脳でのアセチルコリン (ACh) のイメージング(Anal. Bioanal. Chem. 403:1851-1861, 2012)

Fig.5
A

B

図5. latent TGF- binding protein 4 (LTBP-4) による微小線維形成機構の解析(Proc. Natl. Acad. Sci. USA 110:2852-2857, 2013)

はじめに
医学・医療技術の進歩により世界一長寿国となった現在、認知症などの神経系疾患、がん、動脈硬化・糖尿病など代謝性疾患の早期診断と進行予防が喫緊の課題となっています。これらの疾患は生体の正常機能を担う遺伝子の変異、タンパク質やエネルギー物質の変性と蓄積によることが明らかになってきています。また、画像診断、組織診断の進歩により病変部位が可視化され、病気の早期診断と経過観察が可能になっていますが、関与する因子は多様で、治療成績の向上には必ずしもつながっていません。このような医療のニーズや新しい医学に対応するために、関西医科大学は、平成 18 年 1 月に枚方病院、平成 22
年 7 月に香里病院を開院し、平成 25 年 4 月に枚方病院の隣に枚方新学舎が完成
しキャンパスが統合されました。
研究面では、これまで学術フロンティア推進事業の支援を受けた「修復再生医学による神経系難治性疾患の治療に向けた横断的トランスレーショナル研究」（平成 18～22 年）を推進するためにブレインメディカルリサーチセンターを設置し、神経系の「再生」と「自己修復能」に焦点をあて、基礎部門と臨床部門が連携したトランスレーショナル研究を強力に推進する体制を構築してきました。
この事業をさらに継続発展させるために、平成 23 年度文部科学省私立大学戦略的研究基盤形成支援事業「分子イメージングによる体系的病態の解明と診断
治療法の開発」が採択され、平成 23 年 9 月に病態分子イメージングセンターが設立されました。本センターでは、最先端の分子イメージングシステムを駆使して、患者と動物モデルの疾患の病態を分子から個体まで体系的に解明し、課題とする疾患の診断治療法の開発に結びつけることを目的としています。枚方新学舎北棟５階に病態分子イメージングセンターが設置され、領域横断的、戦略的な研究が可能な拠点の基盤形成に着手しています。
平成 25 年 9 月
1Preface
At the present time that Japan is the nation of highest longevity owing to the advanceof medicine and medical technologies, early diagnosis and prevention of the progressionof neurodegenerative diseases such as dementia, cancer, and metabolic disorders such as
atherosclerosis and diabetes are the utmost and urgent matters of interest.

It becomes

evident that these diseases are caused by mutation of genes, deposits of degenerated
proteins, and excessive storage of body fuel, which are indispensable for normal
functions in the body. Although recent progress in imaging and pathological diagnosis
enables us to make early diagnosis and follow-up in many diseases, it does not always
lead to good outcome of treatments due to many factors involving pathogenesis. In
order to meet such needs of medical care and cope with the advance of medicine,Kansai Medical University opened Hirakata hospital in January 2006 and Kori hospitalin July 2010. Hirakata campus next to Hirakata hospital just opened in April 2013 andunified the campus to Hirakata.
To promote the project “Interactive translational research toward the treatment of
intractable neural diseases by restoration and regeneration” (2006－2011) supported by
the Scientific Frontier Promoting Program of the Ministry of Education, Culture, Sports,
Science, and Technology (MEXT) of Japan, we established Brain Medical ResearchCenter and have constructed a system for promotion of translational research between
basic and clinical departments focusing on the abilities of “regeneration” and“self-restoration” in the nervous system
To continue and expand the project, we applied the project “Systematic elucidation ofpathogenesis and development of diagnosis and treatment by molecular imaging” to the
MEXT-supported program for the strategic research foundation at private universities in2011.

Upon its adoption, we established the Molecular Imaging Center of Diseases in

September 2011.

The objectives of the Center are (1) to systematically elucidate

pathogenesis of various diseases by use of animal disease models and patient samples
from molecular to individual levels and (2) to translate the basic research on diseases
into clinical application, by making the most of state-of-the-art imaging systems.

We

have established the Molecular Imaging Center of Diseases on the 5th floor of the northwing in Hirakata new campus and have just started to make it a research base capable ofpursuing interdisciplinary and strategic research.
September 20132

I. 病態分子イメージングセンターの事業概要
１．大学名：関西医科大学
２．研究組織名：病態分子イメージングセンター
３．研究プロジェクト名： 分子イメージングによる体系的病態の解明と診断治
療法の開発
４．研究組織：病態分子イメージングセンターに神経、がん、代謝の３研究部門と支援部門の研究体制を構築し、３部門に部門統括者を配置します。部門統括者は部門内の研究を統括し、部門間の連携と調整を行い、運営会議はプロジェクトの事業計
画、成果報告、予算執行等を審議決定します。
神経部門は神経可塑性・非可塑性、神経変性疾患
がん部門は組織・がん幹細胞の同定、発がん･転移機構
代謝部門は炎症、血管・組織の加齢変化、動脈硬化、糖尿病の病態解明を中心に基礎医学研究から臨床応用研究まで多岐にわたる研究を推進します。
支援部門は本プロジェクトの研究推進と若手研究者の人材育成の支援を行います。
研究成果は学外の委員で構成される評価委員会の評価を受けます。
５．研究施設、設備・装置
《施設》
施設の名称
病態分子
イメージングセンター

整備年度

（千円）

研究施設 研究室等
面積

使用者数 事業経費

補助金

49,552

24,761

135,325

67,662

m2

平成 23 年度
平成 24 年度

数

818

21

62

《装置・設備》
装置・設備の名称（研究装置）
質量顕微鏡
高度細胞機能解析
蛍光イメージングシステム

（千円）

整備年度

型番

平成 23 年度 Imaging-MS for KMU

平成 24 年度

ImageStream imaging
flow cytometer
ArrayScan VTI HCS
reader

事業経費

補助金

76,00038,00072,00036,000（研究設備）
近赤外蛍光イメージャー
Odyssey system
HS オールインワン
蛍光顕微鏡

平成 23 年度

LI-COR9201-00

7,300

4,866

平成 23 年度

BZ-90000

13,4778,980

3

《研究費》（千円）

年度

消耗品費 通信運搬費 旅費交通費 業務委託費

会合費

雑費

修繕費

平成 23 年度

33,704

72

760

4,157

142

333

520

平成 24 年度

40,712

118

1,480

4,443

224

612

5,362

年度

支払手数料

賃貸料

福利費

人件費

教育研究用
機器備品

図書費

合計（円）

平成 23 年度

22

373

0

1,478

12,48886

54,135平成 24 年度

343

0

8

1,276

27,327

63

81,968

６．運営委員会委員

センター長

伊藤

誠二

副センター長

上野

博夫

部門統括者

基礎系

臨床系

神経部門

山田

久夫

日下

博文

がん部門

藤澤

順一

友田

幸一

代謝部門

中邨

智之

岡崎

和一

７．外部評価委員
外部評価委員
神経部門

Prof. Min Zhuo
（University of Toronto, Dept. of Physiology
Editor-in-chief, Molecular Brain, Molecular Pain)

がん部門

山本 雅教授（沖縄科学技術大学院大学 細胞シグナルユニット
前東京大学医科学研究所 癌細胞シグナル分野)

代謝部門

Dr. John Hanover（Lab. Chief, Cell Biochemistry and Biology, National Institute of
Diabetes and Digestive and Kidney Diseases (NIDDK), NIH, USA）

4

８．研究事業者と共同研究者
研究事業者
神経部門

11 部門

がん部門

10 部門

代謝部門

9 部門

共同研究者
龍俊副所長（京都大学 iPS 細胞研究所 臨床応用研究部門）

中畑

瀬藤 光利教授（浜松医科大学 解剖学講座
近江谷克裕部門長（産業技術総合研究所

細胞生物学分野）

バイオメディカル研究部門）

９．研究プロジェクト参加研究者数：事業推進者 30 名

部門区分

事業推進者

課題名
【

神経部門 】

神経機能再生医学山田久夫

神経細胞機能の in situ イメージング

脳構築学

杉本

哲夫

マウス線条体における投射ニューロンのイメージング研究

細胞分子生理学松田博子

病因細胞および分子のイメージング

高次認知脳科学中村加枝

霊長類におけるセロトニン・ドパミンによる認知機能の制御分子生体機能学伊藤誠二

難治性神経疾患の病態解明と診断治療法の開発

神経内科学

日下

博文

筋萎縮性側索硬化症の原因因子の病態に関する研究精神神経科学木下利彦

ミラーニューロンの形態変化：
拡張テンソル画像解析による精神科治療効果判定

脳神経再生医学淺井

昭雄

グリオブラストーマ癌幹細胞のin vivo イメージング法の開発

麻酔科学

新宮

興

樹状細胞における静脈麻酔薬プロポフォールのロイコトリエン産生に及ぼす影響

臨床検査医学髙橋伯夫

高血圧の病態解明のための
脳内内因性ジギタリスの局在分析

救急医学科

中谷

壽男

脊髄損傷に対する細胞治療の実験的・臨床的研究

5

【がん部門 】

幹細胞病理学上野博夫

多色細胞系譜追跡法を用いた幹細胞の維持機構の解析

ウイルス腫瘍学藤澤順一

HTLV-1 感染ヒト化マウスを用いた
ATL 発症過程の解析

幹細胞生物学薗田精昭

ES 細胞様組織幹細胞の同定と分化経路の解明：細胞移植・再生医療への応用

公衆衛生学

西山

利正

組織幹細胞に高発現する遺伝子の機能解析とがん幹細胞との共通性の検討

分子遺伝学部門

木梨達雄

リンパ節のホーミングの分子機構に関する研究

モデル動物部門李成一

遺伝子改変マウス作製による接着分子の可視化
および解析

外科学

權

雅憲

質量顕微鏡法を用いた病理標本解析による
腸管内腔閉塞原因物質の同定と発症機構の解明

皮膚科学

岡本

祐之

質量顕微鏡を用いた皮内メラニンの測定

耳鼻咽喉科学友田

幸一

質量顕微鏡による頭頸部がん関連因子の探索・同定

共同研究講座（幹細胞異常症学）

池原進

難治性神経疾患に対する骨髄内骨髄移植の適用の検討【

代謝部門 】

神経病態薬理学中邨智之

細胞外マトリックス線維形成の加齢変化に関する研究

生体情報部門松田達志

がん幹細胞の機能・分化に関わる
シグナル分子の解明

内科学第一

野村

昌作

ヒト樹状細胞によるさまざまな病態関連応答の解
明と新たな難病治療戦略の開発

内科学第二

塩島

一朗

心臓組織幹細胞の表面マーカー探索

内科学第三

岡崎

和一

分子イメージングによる炎症性消化器疾患のバイオマーカー探索

形成外科学

楠本

健司

皮膚・軟部組織の加齢変化、組織再生における分子イメージングによる病態解明と診断治療への応用

眼科学

高橋

寛二

脈絡膜新生血管・網膜変性・網膜虚血再灌流における分子機構の解明

放射線科学

谷川

昇

固形がんに対する RI 標識抗体による早期イメージング診断剤および治療薬の開発における基礎研究

物理学

影島

賢巳

原子間力顕微鏡による細胞内機能イメージング

6

I. Outline of Molecular Imaging Center of Diseases (MICD)
1． University: Kansai Medical University
2． Organization: Molecular Imaging Center of Diseases
3． Title: Systematic elucidation of pathogenesis and development of diagnosis andtreatment by molecular imaging
4． Management:
The center consists of three research divisions of neuroscience, cancer and metabolic
disorders, each of which is managed by two division heads from basic and clinical fields,
and a supporting division. The division heads integrate research in the division andadjust matters between divisions. The management committee deliberates anddetermines an annual plan, budget and outcome and other pertinent issues of the project.
We promote a wide-range research from basic to clinical medicine focusing on Neuroscience: neural function and neural plasticity, neurodegenerative diseases
 Cancer: identification of tissue & cancer stem cells, mechanisms of
oncogenesis and metastasis
 Metabolic disorders: inflammation, aging in vessels and connective tissues, andpathogenesis of atherosclerosis and diabetes
The supporting division helps the project members to promote research activities andto cultivate young researchers.
Research activities are evaluated by three outside members of advisory board.
5．

Research facilities, apparatuses, and equipment《Facility》(Unit: 1,000)

Name of facility
Molecular Imaging
Center of Diseases

Fiscalyear

Research Number of Number of Operational
facility area laboratories
users
expenses

2011
2012

m2

21

818

62

《Apparatus and equipment》
Name（Apparatus）

Mass spectrometer

Advanced fluorescence imaging
system for cellular function analyses

Subsidy

49,552

24,761

135,325

67,662(Unit: 1,000)

Fiscal year of
installation

Model number
Imaging-MS

2011

for KMU
Image Stream
imaging flow
cytometer

2012

Array Scan VTI HCS
reader7

Operational
Subsidy
expenses
76,00038,00072,00036,000（Equipment）

Infrared imaging system Odyssey
HS All-in-one fluorescencemicroscope

2011

LI-COR9201-00

7,300

4,866

2011

BZ-90000

13,477

8,980《Research expenses》(Unit: 1,000)

Fiscal Experimental Communications
Travel Outsourcing Meeting Miscellaneous Maintenancesupplies& transportation
year
2011

33,704

72

760

4,157

142

333

520

2012

40,712

118

1,480

4,443

224

612

5,362

WelfarePersonnel

Machinery
appliance

FiscalCommission
year

Lease

Book

Total (Yen)

2011

22

373

0

1,478

12,48886

54,135

2012

343

0

8

1,276

27,327

63

81,968

6． Committee members

Director

Seiji Ito

Vice-director

Hiroo Ueno

Division head

Basic

Clinical

Neuroscience

Hisao Yamada

Hirofumi Kusaka

CancerJun-ichi Fujisawa

Koichi Tomoda

Metabolic Disorders

Tomoyuki Nakamura

Kazuichi Okazaki

7． Advisory board members
Advisory board
Prof. Min Zhuo
Division of Neuroscience(Dept. of Physiology, University of Toronto,
Molecular Brain, Molecular Pain, Editor-in-chief)
Prof. Tadashi Yamamoto

Division of Cancer(Cell Signal Unit, Okinawa Institute of Science and Technology
Graduate University)
Dr. John Hanover

Division of Metabolic Disorders(Lab. Chief, Cell Biochemistry and Biology, National Instituteof Diabetes and Digestive and Kidney Diseases, NIH, USA)8

8． Core members of the CenterCore members
Division of Neuroscience

11 departments

Division of Cancer

10 departments

Division of Metabolic Disorders

9 departments
Cooperative members

Vice President Tatsuyosi Nakahata（Center for iPS Cell Research and Application, Kyoto University）
Prof. Mitsutoshi Setou（Cell Biology & Anatomy, Hamamatsu University School of Medicine）
Dr. Yoshihiro Ohmiya（Bioproduction Research Institute, National Institute of Advanced Industrial Science andTechnology）

9． Research projects
DivisionCorememberTitle of research project【

Neuroscience 】

Regenerative Neural
Medicine

Hisao
YamadaIn situ imaging of neural cells and their function

Anatomy and Brain
Science

TetsuoSugimoto

Imaging of projection neurons of mouse striatum

Cellular andMolecular Physiology

HirokoMatsudaImaging of disease-related molecules and cells

Cognitive
Neuroscience

Kae
Nakamura

The role of serotonin and dopamine for cognitive processes
in the primate

Molecular andFunctional Biology

Seiji Ito

Elucidation of pathogenesis and development of diagnosis
and treatment of intractable neural diseases

Neurology

Hirofumi
KusakaStudy on pathogenic factors in amyotrophic lateralsclerosis

Neuropsychiatry

ToshihikoKinoshita

Elucidation of pathogenesis of psychiatric disorder-Motor
related white abnormalities in patients with schizophrenia

Neurosurgery

Akio Asai

Development of in vivo imaging of cancer stem cells inglioblastoma

Medical Control ofStress Response

Koh Shingu

Effect of intravenous anesthetic propofol on leukotriene
production in murine dendritic cells

Clinical Sciences andLaboratory Medicine

Hakuo
Takahashi

brain, and the pathophysiological roles in hypertension

Spinal Cord
Regeneration

ToshioNakatani

Basic and clinical studies on cell treatment for complete
spinal cord injury

Analysis of localization of endogenous digitalis in the9【Cancer 】

Stem Cell Pathology

Hiroo Ueno

Analyses of dynamics of intestinal stem cells by
multi-color lineage tracing method

Viral Oncology

Jun-ichiFujisawa

Analysis of ATL development in HTLV-1 infected
humanized mouse model

Stem Cell Biology

Yoshiaki
SonodaIdentification of ES cell-like tissue committed stem cells
and elucidation of their differentiation pathway: An
application to cell therapy and regenerative medicine

Public Health andInternational HealthToshimasa
Nishiyama

Functional analysis of overexpressing genes in tissue stem
cells and searching the relationship with cancer stem cells

Molecular Genetics

TatsuoKinashiMolecular mechanism of lymphocyte trafficking

Model AnimalSung-il Lee

Visualization and analysis of adhesion molecules using
genetically modified animal

Surgery

A-Hon
Kwon

Identification of causative material of small-bowel
obstruction from histopathological sections by
MALDI-imaging mass spectrometry

Dermatology

HiroyukiOkamotoAnalysis of UV-induced molecules related to melanoma

Otolaryngology

Koichi
TomodaSearch and Identification of head and neck cancer-related
factors by imaging mass spectrometry

Stem Cell Disorders

SusumuIkeharaExamination of application of IBM-BMT to intractablediseases of the nervous system【 Metabolic Disorders 】

Neuropharmacology

TomoyukiNakamura

Study of fibril formation in the extracellular matrix

Cell Signaling

SatoshiMatsudaAnalysis of signal transduction molecules involved inproliferation, development and stemness

Internal Medicine 1

Shosaku
NomuraDevelopment of novel therapeutic strategy and elucidationof functional basis for intractable diseases by humandendritic cell researchInternal Medicine 2

IchiroShiojima

Identification of cardiac stem cell-specific surface antigens

Internal Medicine 3

KazuichiOkazakiA molecular imaging study of identifying biomarkers in
inflammatory gastroenterological disorders

Plastic andReconstructive
Surgery

KenjiKusumoto

Clarification of skin and soft tissue aging and tissue
regeneration and application in diagnosis and treatmentusing molecular imaging

Ophthalmology

Kanji
Takahashi

Elucidation of molecular mechanism of choroidal
neovascularization, retinal degeneration and retinal
ischemia-reperfusion injury

Radiology

NoboruTanigawa

Development for diagnostic agent and the curative drugsby the RI labeled antibody for the solid tumors

Physics

MasamiKageshima

Cellular function imaging by atomic force microscopy
10II. 研究活動の進捗状況・研究成果の要約
１．研究の進捗状況
病態分子イメージングセンターの研究成果は Nat. Cell Biol., Nat. Commun.,
Neuron, Proc. Natl. Acad. Sci. USA, J. Biol. Chem., J. Neurosci., J. Immunol., J.
Gastroenterol. をはじめとして欧文雑誌に 150 編以上発表し、滝井学舎の基礎系の事業推進者の研究は順調に進捗し、達成度は満足すべきものである。臨床系事業推進者の多くは枚方キャンパスでの診療に従事していたことから、実験室がある滝井学舎への物理的乖離（京阪電車で 40－50 分）、新医師研修必修化による医師不足、大学院離れによる研究スタッフの減少により、達成度は必ずしも満足するべきものではない。2013 年 4 月に枚方新学舎が開校し、病態分子イメージングセンターが北棟 5 階に開設されたことにより、本プロジェクトの参加者はすべて徒歩 5 分以内で同センターにアクセスできるようになり、物理的隘路は解消された。本プロジェクトの 3 年目以降は、基礎系講座、臨床系講座
間の共同研究、トランスレーショナル研究が進展するものと期待する。事業推進者 30 名の研究活動の進捗状況を要約するが、全事業推進者の詳細な研究活動
は平成 23 年度研究成果報告、平成 24 年度研究成果報告として後掲した。
１）幹細胞の同定
上野（幹細胞病理学）は自身で確立した多細胞系譜追跡法（Nature 464:549-553,
2010）を用いて、舌の毛状乳頭小窩基底部に長期間生存する Bmi1 陽性の幹細胞が 1 個存在し、角化上皮細胞の維持再生に関与することを報告した（Nature Cell
Biol. 15:511-518, 2013）。伊藤ら（生体分子機能学）が同定したショウジョウバ
エの卵形成に関与する遺伝子 ovo のマウス相同遺伝子 Ovol2 (Movo) は Zn フィ
ンガー型転写因子で ES 細胞に発現する。今回、成体では精巣特異的に発現し、作製した抗体で精母細胞のパキテン期の XY 体に発現していることを明らかにして、精子形成に関与する可能性を示した（J. Androl. 33:277-286, 2012）。山田ら（神経機能再生医学）は側脳室周囲の神経前駆細胞のニューロン新生は厳密に調節されており、その修正には強い刺激が必要であることを示した。薗田（幹細胞生物学）は骨組織由来の微小幹細胞の効率的な分離方法を開発した（特願2012-220648）。神田（公衆衛生学）と杉本（脳構築学）は下垂体と聴覚伝導路の組織幹細胞の同定を行った。塩島ら（内科学第 2）はヒト心臓組織幹細胞を効
率よく末梢血中に動員する方法を確立し、その機序を明らかにした。
２）分子イメージング
木梨（分子遺伝学）はリンパ節スライス法による二光子イメージングを樹立
し、T 細胞の組織内高速移動に LFA-1/ICAM-1 を介する接着が必要であることを明らかにした。さらに、Mst1 ノックアウトマウスで制御性 T 細胞の抗原依存的抑制作用が障害されていたことから、Mst1 による胸腺上皮細胞の接着制御が自己抗原による負の選択に必要であることを示した（Nat. Commun. 3:1098, 2012）。
質量顕微鏡が神経化学の領域において、小分子化合物の分布のイメージングに有用なツールとなっている。矢尾（生体分子機能学）は神経伝達物質の１つ
アセチルコリン(ACh)のイメージングに初めて成功した。ACh のラット脳での分布は、その分解酵素アセチルコリンエステラーゼの分布と一致していた（Anal.
Bioanal. Chem. 403:1851-1861, 2012）。従来の分解酵素の代謝産物の測定と異なり、大脳皮質や運動ニューロンのシナプス終末の vesicle の ACh を測定することから、
剖検脳や動物モデルの病態解析に有用と期待される。權（外科学）は小腸で通11

過障害を起こした物質がモチであることを質量顕微鏡で明らかにした。日下（神経内科学）は筋萎縮性側索硬化症患者の剖検脳の封入体の解析、高橋（臨床検査医学）内因性ジギタリス様物質の解析を進めている。杉本（脳構築学）は新しいイメージング法を用いて、線条体外側部のニューロンの特徴を明らかにした。影島（物理学）は軟 X 線顕微鏡や原子間力顕微鏡でタンパクから細胞のイ
メージング法の開発に取り組んでいる。中村、磯田（高次認知脳科学）らは、サルを用いて報酬、罰など高次脳機能の個体での情報処理と社会的相互作用を解析している。
３）臨床応用に向けた病態の解明
細胞外マトリクスの 1 つである微小線維は肺、大動脈、皮膚など伸縮性のある組織の弾性線維形成の足場となる一方、眼球の毛様体小体では微小線維そのものが水晶体を毛様体筋に保持する。中邨、赤間（分子薬理学）は latent TGF-
binding protein 4 (LTBP-4) が fibulin-5 と会合して弾性線維形成に関与すること、
弾性線維再生の治療標的になる可能性を示した（Proc. Natl. Acad. Sci. USA
110:2852-2857, 2013）。さらに、中邨らが作製した LTBP-2 の遺伝子欠損マウス
は毛様体小体の線維形成が障害されており、水晶体脱臼、緑内障などヒトの眼疾患のモデル動物となることを示した。下條（分子生体機能学）は京都大学 iPS細胞研究所との共同研究で作製したハンチントン病患者と健常人の iPS 細胞を用いた病態解明を開始した。藤澤（ウイルス腫瘍学）は HTLV-1 感染ヒト化マ
ウスがその発症予防薬の評価系に有効であることを示した。松田（生体情報部
門）は種々のがん組織で異常がみられる mTORC1 経路のアダプタータンパク
Raptor の細胞系譜特異的欠損マウスの樹立し、個体レベルでの薬剤の検証に成功した。野村と伊藤（内科学第 1）は、アレルギーの機序に樹状細胞だけでなく、血小板の役割を解明した。岡崎（内科学 3）は IgG4 陽性細胞が膵癌と IgG4 関連
自己免疫性膵炎との鑑別のバイオマーカーとなる可能性を示した。楠本（形成外科学）は病的創傷治癒、組織再生の基礎研究を行っている。池原（幹細胞異常症学）は自身が開発した骨髄内骨髄移植法と胸腺移植の組合せが自己免疫疾患や加齢に伴う症状の治療に有効であることを示唆した。中谷ら（脊髄再生医学）は脊髄損傷の治療に向けて骨髄細胞の髄液内投与の臨床試験を行い、その安全性を確かめた。
2. 滝井キャンパスから枚方キャンパスへの引越し
病態分子イメージングセンターは 2013 年 3 月に滝井キャンパスから枚方キャンパス北棟 5 階に移転した。基礎社会医学系講座、臨床系講座、一般教養の教室・研究室は中央棟の 10‐12、8‐9、2 階にあり、病態分子イメージングセン
ターには数分で行くことができる。共焦点顕微鏡、蛍光顕微鏡など従来からある多くの顕微鏡に加え、オールインワン顕微鏡は顕微鏡室（図 1 左上）に配置
され、多光子励起顕微鏡に加え、新たに質量顕微鏡の部屋（図 1 左下）が設置された。RI イメージング室は基礎研究を臨床応用するために設置された。臨床
綜合研究施設は日本の医科大学ではユニークな施設で臨床系講座の枠を超えた共同研究が促進されることを期待している。研究を円滑、効率的に進めるために、管理運営を支援する技師を臨床系綜合研究施設に置いた。

12

図 1．病態分子イメージングセンター（枚方キャンパス北棟 5 階）

３．研究活性化のための山下学長の 3 つの提案と講演会、シンポジウムの開催
慶応義塾大学医学部岡野栄之教授（平成 24 年 3 月 21 日）と理化学研究所発
生・再生化学総合研究センター笹井芳樹グループディレクター（平成 25 年 3 月
22 日）の特別講演会をはじめ、この 2 年間で招待研究者による講演会を 20 回以上開催した。共同利用研究施設による機器説明会、利用説明会を大学院総合講
義の中に組み込み若手研究者の育成支援を行った。枚方学舎での講座の垣根を越えた共同研究を推進するために、山下敏夫学長は 3 つの提案 １）再生医療
コンソーシアム、２）がん関連コンソーシアム、３）研究トークランチを行った。再生医療コンソーシアムは基礎研究から臨床研究、臨床応用への橋渡し、がん関連コンソーシアムはがんの診断、治療、合併症に関する臨床主体のグループ研究、３）研究トークランチは 1 ヶ月に 1 回基礎医学系講座の研究紹介、プログレスレポートで基礎社会医学系講座間の意思疎通を図る企画で、平成 25
年度から実施している。

図 2．病態分子イメージングセンターと 4 つの新プロジェクト

13II. Activities and progress in research of MICD
1. Research activities
The outcome of Molecular Imaging Center of Diseases (MICD) has been publishedin more than 150 international journals including Nat. Cell Biol., Nat. Commun.,
Neuron, Proc. Natl. Acad. Sci. USA, J. Biol. Chem., J. Neurosci., J. Immunol., J.
Gastroenterol. and basic researchers in Takii campus have made favorable progress andachieved satisfactory accomplishments during the first 2 years of the project. On theother hand, most of core members in clinical fields working at Hirakata hospital havenot always achieved satisfactory accomplishments due to the distance (40-50 minutes’
ride of train) between Hirakata and Takii campus where experimental facilities had been
located, shortage of clinical staffs and decrease in graduate students. Upon opening of
Hirakata campus this April, researchers of all departments can access to MICD within a
few minutes’ walk. We expect that research in individual departments will expand tocollaborative research beyond departments, translational research from basic to clinicalmedicine such as early diagnosis and treatment in the latter 3 years of this project.
This section picks up topics of research activities of 30 core members in MICD.
Please refer to a list of publications of all departments and annual reports 2011 and 2012
for further details.
1) Identification of stem cells
Ueno (Stem Cell Pathology) showed that stem cells positive for Bmi1, keratin 14
and keratin 5 are present in the base but not at the very bottom of the interpapillary pit
and that one stem cell per interpapillary pit survives long-term using a multicolor
lineage tracing method established by Ueno (Nature 464:549–553, 2010; Nat. Cell Biol.
15:511-518, 2013). A mouse homologue of Drosophila ovo, Ovol2 identified by Ito’s
group (Molecular and Functional Biology) is a zinc finger transcriptional factor andexpressed in embryonic stem cells. Ito revealed that Ovol2 expression is restricted to
the XY body in spermatocytes at the pachytene stage and is suggested to play animportant role in spermatogenesis (J. Androl. 33, 277-286, 2012).
Yamada (Regenerative Neural Medicine) showed that neuronal precursors in thesubventricular zone (SVZ) are tightly regulated and require stimuli to be modified
stronger than those in the subgranular zone (SGZ). Sonoda (Stem Cell Biology)
developed a highly effective isolation method of mouse bone-derived very small
embryonic-like stem cell. Kanda (Public Health) and Sugimoto (Anatomy and Brain
Science) focused on tissue stem cells in the pituitary and auditory pathway. Shiojima(Internal Medicine 2) developed a method to effectively mobilize cardiac stem cells into
peripheral blood via upregulation of hepatic growth factor.
2) Molecular imaging
With two-photon imaging using lymph node tissues, Kinashi (Molecular Genetics)
14

revealed that high-speed interstitial migration of T cells requires adhesion throughLFA-1 and ICAM-1. Since thymocyte selection is impaired in Mst1-/- mice, Mst1 hasa key role in regulating self-antigen recognition by thymocytes in the medulla (Nat.
Commun. 3:1098, 2012).
Metabolite distribution imaging via imaging mass spectrometry (IMS) is anincreasingly utilized tool in the field of neurochemistry. Yao (Molecular and Functional
Biology) first succeeded in imaging of ACh by IMS, showing that its distribution iscoincident with that of the degrading enzyme ACh esterase in rat brain (Anal. Bioanal.
Chem. 403:1851-1861, 2012). The detection of ACh by IMS will be useful to analyze
animal models and postmortem brain samples of patients with neurodegenerative
disorders such Alzheimer’s disease. Kwon (Surgery) succeeded in the identification of
the obstructing material in small intestine obstruction by IMS. Kusaka (Neurology)
started analysis of intracytoplasmic inclusions of human brain section suffered withneurodegenerative diseases such as amyotrophic lateral sclerosis and Takahashi(Clinical Sciences and Laboratory Medicine), endogenous digitalis, by IMS. Sugimoto
characterized neurons in the lateral region of striatum by a novel imaging method.
Atomic force microscopy (AFM) has gained recognition as an imaging tool for surfacesof various materials ranging from solids to soft biological materials. Kageshima(Physics) has tried to visualize proteins and cells by AFM. Nakamura & Isoda(Cognitive Neuroscience) have analyzed information processing of higher brain
functions such as reward, punishment and social mentality in the primate.
3) Elucidation of pathogenesis toward clinical application
While microfibrils, an extracellular matrix, provide a scaffold for elastic tissues
such as lung, aorta and skin, they themselves connect lens with ciliary muscle in theciliary body of the eye. Nakamura & Akama (Neuropharmacology) showed that latent
TGF-β binding protein 4 (LTBP-4) potentiates formation of elastic fibers throughinteracting with fibulin-5, a tropoelastin-binding protein necessary for elastogenesis(Proc. Natl. Acad. Sci. 110: 2852-2857, 2013). They also found that LTBP-2
knockout mice impaired the formation of ciliary body, suggesting that they are an ideal
model animal for congenital lens luxation, ectopia lentis and glaucoma in human.
Shimojo (Molecular and Functional Biology) started to study the pathogenesis ofHuntington’s disease using iPS cells prepared from healthy and patient cells incollaboration with Center for iPS Cell Research and Application (CiRA). Fujisawa(Viral Oncology) showed that HTLV-infected humanized mice are useful to evaluate
preventives for HTLV.
Matsuda (Cell Signaling) established mTORC1
signal-deficient mice lacking Raptor specifically in dendritic cell lineage in the intestineand succeeded in evaluation of drugs in vivo. Nomura & Ito (Internal Medicine 1)
provided new insights of platelet function into the possible mechanism of dendritic
cell-mediated allergic response. Okazaki (Internal Medicine 3) demonstrated a15

possibility that IgG4-positive cells can be a biomarker for differential diagnosis of
autoimmune pancreatitis and pancreas cancer. Kusumoto (Plastic and Reconstructive
Surgery) has been engaged in basic research of wound healing and regenerative
medicine toward treatment and reconstruction. Ikehara (Stem Cell Disorders) who
established intra-bone marrow-bone marrow transplantation (IBM-BMT) suggested thatboth IBM-BMT and thymus transplantation can be effective interventions in thetreatment of autoimmune diseases and age-associated symptoms. Nakatani (Spinal
Cord Regeneration) confirmed safety for the treatment of spinal cord injury byadministration of autologous bone marrow stromal cells into cerebrospinal fluid.

2. Moving from Takii to Hirakata campus in March 2013
MICD was moved to 5th floor of the north wing of Hirakata new campus in March
2013. Laboratories belong to individual departments are located on 2 and 8-12 floorsof the central building of Hirakata campus. Many conventional microscopes as well as
all-in-one fluorescence microscope are gathered in the microscopic room (Fig. 1, upper
left) and two-photon microscopy and imaging mass spectrometry are set in respective
rooms (lower left). RI imaging room is installed to translate basic research to clinicalapplication (upper right). The idea of Clinical Central Research Laboratory (CCRL)(lower middle and right) is unique to Japanese medical school and will promote thecollaborative research beyond clinical departments. To promote research smoothlyand efficiently, a technical assistant is arranged for management and maintenance of
CCRL.

Fig. 1 Map of MICD on 5th floor of the north wing of Hirakata campus

3. Three projects proposed by President Yamashita in April 2013
More than twenty lectures were held by invited speakers and technical seminars
were also held for researchers. To promote collaborative research beyond departments,
President Toshio Yamashita has proposed three projects, 1) regenerative medicine
consortium which accelerates translational research from basic to clinical research and16will extend to the treatment by regenerative medicine, 2) cancer-oriented consortium
forms a group research concerning diagnosis, treatments and complications of cancer inclinic, and 3) research talk at lunch where an outline of research and experimental data
are presented and discussed mainly by researchers of departments belonging to basic
medicine at lunch once a month. In addition to research talk at lunch, we also started a
training course of physician scientists for medical students in liaison with Nara Medical
University and Osaka Medical College to cultivate young basic researchers.

Fig. 2 Management of MICD and 4 new projects of MICD

17

III.

原著論文リスト

List of original articles

Division of Neuroscience 神経部門
Regenerative Neural Medicine 神経機能再生医学

1.
2.
3.
4.
5.
6.
7.

Cui, Y., Li, Q-H., Yamada, H., Watanabe, Y. and Kataoka, Y. Chronic degeneration of dorsal
Raphe serotonergic neurons modulates cortical spreading depression: a possible
pathophysiology of migraine. J. Neurosci. Res. 91, 737–744, 2013.
Hirahara, Y., Matsuda, K.I., Liu, Y.F., Yamada, H., Kawata, M. and Boggs, J.M. 17β-Estradiol
and 17α-Estradiol induce rapid changes in cytoskeletal organization in cultured
oligodendrocytes. Neuroscience 235, 187-199, 2013.
Hirahara, Y., Matsuda, K.I., Yamada, H., Saitou, A., Morisaki, S., Takanami, K., Boggs, J.M.
and Kawata, M. G protein-coupled receptor 30 contributes to improved remyelination after
cuprizone-induced demyelination. Glia 61, 420-431, 2013.
Kimura, T., Jiang, S., Nishizawa, M., Yoshigai, E., Hashimoto, I., Nishikawa, M., Okumura, T.
and Yamada, H. Stabilization of human interferon-α1 mRNA by its antisense RNA. Cell Mol.
Life Sci. 70, 1451-1467, 2013.
Mori, T., Wakabayashi, T., Hirahara, Y., Takamori, Y., Koike, T., Kurokawa, K. and Yamada, H.
Differential responses of endogenous adult mouse neural precursors to excess neuronal
excitation. Eur. J. Neurosci. 36, 3184-3193, 2012.
Wakabayashi, T., Kosaka, J., Mori, T. and Yamada, H. Prolonged expression of puma incholinergic amacrine cells during the development of rat retina. J. Histochem. Cytochem. 60,
777-788, 2012.
Wakabayashi, T., Mori, T., Hirahara, Y., Koike, T., Kubota, Y., Takamori, Y. and Yamada, H.
Nuclear lamins are differentially expressed in retinal neurons of the adult rat retina. Histochem.
Cell Biol. 136, 427-436, 2011.

Anatomy and Brain Science 脳構築学

1.
2.
3.
4.

Maruyama, M., Yamashita, Y., Kase, M., Trifonov, S. and Sugimoto, T. Lineage-specificpurification of neural stem/progenitor cells from differentiated mouse induced pluripotent stem
cells. Stem Cells Transl. Med. 2, 420-433, 2013.
Horie, A., Fujiwara, H., Sato, Y., Suginami, K., Matsumoto, H., Maruyama, M., Konishi, I. and
Hattori, A. Laeverin/aminopeptidase Q induces trophoblast invasion during human earlyplacentation. Hum. Reprod. 27, 1267-1276, 2012.
Trifonov, S., Houtani, T., Kase, M., Toida, K., Maruyama, M., Yamashita, Y., Shimizu, J. and
Sugimoto, T. Lateral regions of the rodent striatum reveal elevated glutamate decarboxylase 1mRNA expression in medium-sized projection neurons. Eur. J. Neurosci. 35, 711-722, 2012.
Hamada, S., Trifonov, S., Houtani, T., Kase, M., Yamashita, Y., Baba, K., Shimizu, J.,
Maruyama, M., Tomoda, K., Yamashita, T. and Sugimoto, T. Cholinergic fibers and muscarinic
receptors in the central auditory system. J. Kansai Med. Univ. 63, 7-9, 2012.

Cellular and Molecular Physiology 細胞分子生理学

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Okada, M., Matsuda, H. and Okimura, Y. Lentiviral and Moloney retroviral expression of green
fluorescent protein in somatotrophs in vivo. PLoS One, 8, e54437(1-11), 2013.
Hayashi, M., Wang ,J., Hede, S.E. and Novak, I. An intermediate-conductance Ca2+-activated
K+ channel is important for secretion in pancreatic duct cells. Am. J. Physiol. Cell Physiol. 303,
C151−C159, 2012.

Cognitive Neuroscience 高次認知脳科学

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Isoda, M. Recent advances in social neuroscience research using macaques. Brain and Nerve 65,679-686, 2013.
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Isoda, M. Using saccades as a probe of higher brain functions. Neuro Ophthalmol. Jpn. in press,
2013.
McCairn, K.W., Iriki, A. and Isoda, M. Deep brain stimulation reduces tic-related neuralactivity via temporal locking with stimulus pulses. J. Neurosci. 33, 6581-6593, 2013.
McCairn, K.W., Iriki, A. and Isoda, M. Global dysrhythmia of cerebro-basal ganglia-cerebellar
networks underlies motor tics following striatal disinhibition. J. Neurosci. 33, 697-708, 2013.
Yamada, H., Inokawa, H., Matsumoto, N., Ueda, Y., Enomoto, K. and Kimura, M. Coding of
the long-term value of multiple future rewards in the primate striatum. J. Neurophysiol. 109,
1140-1151, 2013.
Yoshida, K., Saito, N., Iriki, A. and Isoda, M. Social error monitoring in macaque frontal cortex.
Nat. Neurosci. 15, 1307-1312, 2012.
Nakamura, K., Santos, G.S., Matsuzaki, R. and Nakahara, H. Differential Reward Coding in the
Subdivisions of the Primate Caudate during an Oculomotor Task. J. Neurosci. 32, 15963-15982,
2012.
Okada, K., Nakamura, K. and Kobayashi, Y. A neural correlate of predicted and actual
reward-value information in monkey pedunculopontine tegmental and dorsal raphe nucleus
during saccade tasks. Neural Plasticity 579840, 1-21, 2011.

Molecular and Functional Biology 分子生体機能学
1.

Unezaki, S., Sasaki, A., Mabuchi, T., Matsumura, S., Katano, T., Nishio, N., Andoh, T.
Nakazawa, T. Yamamoto, T., Nakatsuka, T., Kuraishi, Y. and Ito, S. Involvement of Tyr1472
phosphorylation of NMDA receptor NR2B subunit in postherpetic neuralgia in model mice.
Mol. Pain 8, 59 (1-13), 2012.
2. Takagi, H., Setou, M., Ito, S. and Yao, I. SCRAPPER regulates the thresholds of long-term
potentiation/depression, the bidirectional synaptic plasticity in hippocampal CA3-CA1 synapses.
Neural Plast. 2012, 352829, 2012.
3. Yamada, M., Yao, I., Hayasaka, T., Ushijima, M., Matsuura, M., Takada, H., Shikata, N., Setou,
M., Kwon, AH. and Ito, S. Identification of oligosaccharides from histopathological sections by
MALDI imaging mass spectrometry. Anal. Bioanal. Chem. 402, 1921-1930, 2012.
4. Okuda-Ashitaka, E., Minami, T., Tsubouchi, S., Kiyonari, H., Iwamatsu, A., Noda, T., Handa,
H. and Ito, S. Identification of NIPSNAP1 as a nocistatin-interacting protein involving paintransmission. J Biol Chem. 287, 10403-10413, 2012.
5. Sugiura, Y., Zaima, N., Setou, M., Ito, S. and Yao, I. Visualization of acetylcholine distribution
in central nervous system tissue sections by tandem imaging mass spectrometry. Anal. Bioanal.
Chem. 403, 1851-1861, 2012.
6. Araki, Y., Kaibori, M., Matsumura, S., Kwon, AH. and Ito, S. Novel strategy for the control of
postoperative pain: long-lasting effect of an implanted analgesic hydrogel in a rat model of
postoperative pain. Anesth. Analg. 114, 1338-1345, 2012.
7. Lu, J., Katano, T., Nishimura, W., Fujiwara, S., Miyazaki, S., Okasaki, I., Aritake, K., Urade, Y.,
Minami, T. and Ito S. Proteomic analysis of cerebrospinal fluid before and after intrathecal
injection of steroid into patients with postherpetic pain. Proteomics 12, 3105-3112, 2012.
8. Hayasaka, T., Goto-Inoue, N., Ushijima, M., Yao, I., Yuba-Kubo, A., Wakui, M., Kajihara, S.,
Matsuura, M. and Setou, M. Development of imaging mass spectrometry (IMS) dataset extract
software. Anal. Bioanal. Chem. 401, 183-193, 2011.
9. Shimojo, M. RE1-silencing transcription factor (REST) and REST-interacting LIM domain
protein (RILP) affect P19CL6 differentiation. Genes Cells 16, 90-100, 2011.
10. Ohnaka, M., Okuda-Ashitaka, E., Kaneko, S., Ando, A., Maeda, M., Furuta, K., Suzuki, M.,
Takahashi, K., and Ito, S. Induction of arginase II mRNA by nitric oxide using an in vitro
model of gyrate atrophy of choroid and retina. Invest. Ophthalmol. Vis. Sci. 52, 1493-1500,
2011.
11. Katano, T., Nakazawa, T., Nakatsuka, T., Watanabe, M., Yamamoto, T. and Ito, S.
Involvement of spinal phosphorylation cascade of Tyr1472-NR2B, Thr286-CaMKII, andSer831-GluR1 in neuropathic pain. Neuropharmacology 60, 609-616, 2011.
12. Yao, I., Takao, K., Miyakawa, T., Ito, S. and Setou, M. Synaptic E3 ligase SCRAPPER incontextual fear conditioning; extensive behavioral phenotyping of Scrapper heterozygote andoverexpressing mutant mice. PLoS ONE 6, e17317, 2011.
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13. Kanazawa, M., Furuta, K., Doi, H., Mori, T., Minami, T., Ito, S. and Suzuki, M. Synthesis of anacromelic acid A analog-based 11C-labeled PET tracer for exploration of the site of action ofacromelic acid A in allodynia induction. Bioorgan. Med. Chem. Lett. 21, 2017-2020, 2011.
14. Chizaki, R., Yao, I., Katano, T., Matsuda, K. and Ito, S. Restricted expression of Ovol2/MOVO
in XY body of mouse spermatocytes at the late pachytene stage. J. Androl. 33, 2, 2011.
15. Lu, J., Katano, T., Okuda-Ashitaka, E., Oishi, Y., Urade, S. and Ito, S. Rapid S-nitrosylation ofactin by NO-generating donors and in inflammatory pain model mice. Mol. Pain 7, 101, 2011.
16. Okazaki, T., Otani, H., Shimazu, T., Yoshioka, K., Fujita, M., Katano, T., Ito, S. and Iwasaka,
T. Reversal of inducible nitric oxide synthase uncoupling unmasks tolerance toischemia/reperfusion injury in the diabetic rat heart. J. Mol. Cell Cardiol. 50, 534-544, 2011.
17. Nishida, K., Nakayama, K., Yoshimura, S. and Murakami, F. Role of Neph2 in pontine nuclei
formation in the developing hindbrain. Mol. Cell. Neurosci. 46, 662-670, 2011.
18. Nishiyori, M., Uchida, H., Nagai, J., Araki, K., Mukae, T., Kishioka, S. and Ueda, H.
Permanent relief from intermittent cold stress-induced fibromyalgia-like abnormal pain byrepeated intrathecal administration of antidepressants. Mol. Pain 7, 69, 2011.
Neurology 神経内科学

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2.
3.

4.

5.
6.

7.

Asayama, S., Wate, R., Kaneko, S., Asayama, T., Oki, M., Tsuge, A., Nagashima, M., Morita, J.,
Nakamura, S., Nakamura, M., Nishii, M., Fujita, K., Saito, A., Nakano, S., Ito, H. and Kusaka,
H. Levodopa challenge test and 123I-metaiodobenzylguanidine scintigraphy for diagnosing
Parkinson's disease. Acta Neurol. Scand. 128, 160-165, 2013.
Nakamura, M., Kaneko, S., Shinde, A., Morita, J., Fujita, K., Nakano, S., and Kusaka, H.
Prednisolone-sparing effect of cyclosporin A therapy for very elderly patients with myasthenia
gravis. Neuromuscul. Disord. 23, 176-179, 2013.
Nakamura, M., Kaneko, S., Wate, R., Asayama, S., Nakamura, Y., Fujita, K., Ito, H., andKusaka, H. Regionally different immunoreactivity for Smurf2 and pSmad2/3 in
TDP-43-positive inclusions of amyotrophic lateral sclerosis. Neuropathol. Appl. Neurobiol. inpress, 2012.
Nakamura, M., Kaneko, S., Ito, H., Jiang, S., Fujita, K., Wate, R., Nakano, S., Fujisawa, JI. andKusaka, H. Activation of transforming growth factor-β/Smad signaling reduces aggregate
formation of mislocalized TAR DNA-binding protein-43. Neurodegener. Dis. 11, 182-193,
2013.
Nakamura, S., Nakano, S., Nishii, M., Kaneko, S., and Kusaka, H. Localization of
O-GlcNAc-modified proteins in neuromuscular diseases. Med. Mol. Morphol. 45, 86-90, 2012.
Ito, H., Nakamura, M., Komure, O., Ayaki, T., Wate, R., Maruyama, H., Nakamura, Y., Fujita,
K., Kaneko, S., Okamoto, Y., Ihara, M., Konishi, T., Ogasawara, K., Hirano, A., Kusaka, H.,
Kaji, R., Takahashi, R. and Kawakami, H. Clinicopathologic study on an ALS family with aheterozygous E478G optineurin mutation. Acta Neuropathol. 122, 223-229, 2011.
Honjo, Y., Kaneko, S., Ito, H., Horibe, T., Nagashima, M., Nakamura, M., Fujita, K., Takahashi,R., Kusaka, H., and Kawakami, K. Protein disulfide isomerase-immunopositive inclusions inpatients with amyotrophic lateral sclerosis. Amyotroph. Lateral Scler. 12, 444-450, 2011.

Neuropsychiatry 精神神経科学

1.

2.

Nishida, K., Morishima, Y., Yoshimura, M., Isotani, T., Irisawa, S., Jann, K., Dierks, T., Strik,W., Kinoshita, T. and Koenig, T. EEG microstates associated with salience and frontoparietal
networks in frontotemporal dementia, schizophrenia and Alzheimer's disease. Clin.
Neurophysiol. 124, 1106-1114, 2013.
Kato, M., Nonen, S., Serretti, A., Tetsuo, S., Takekita, Y., Azuma, J. and Kinoshita, T.
5-HTTLPR rs25531A > G differentially influence paroxetine and fluvoxamine antidepressantefficacy: a randomized controlled trial. J. Clin. Psychopharmacol. 33, 131-132, 2013.

Anesthesiology 麻酔科学

1.

Inada, T., Ueshima, H., and Shingu, K. Intravenous anesthetic propofol suppresses leukotriene
production in murine dendritic cells. J. Immunotoxicol. 10, 262-269, 2012.
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Kubo, K., Inada, T., and Shingu, K. Possible role of propofol's cyclooxygenase-inhibiting
property in alleviating dopaminergic neuronal loss in the substantia nigra in an MPTP-induced
murine model of Parkinson's disease. Brain Res. 1387, 125-133, 2011.
Inada, T., Kubo, K., Ueshima, H. and Shingu, K. Intravenous anesthetic propofol suppresses
prostaglandin E2 production in murine dendritic cells. J. Immunotoxicol. 8, 359-366, 2011.
Inada, T., Kubo, K. and Shingu K. Possible link between cyclooxygenase-inhibiting andantitumor properties of propofol. J. Anesth. 25, 569-575, 2011.

Clinical Sciences and Laboratory Medicine 臨床検査医学

1.
2.

3.
4.

Takahashi, H. Upregulation of the renin-angiotensin-aldosterone-ouabain system in the brain is
the core mechanism in the genesis of all types of hypertension. Int. J. Hypertens. 2012, 242728,
2012.
Takahashi, H., Yoshika, M., Komiyama, Y., and Nishimura, M. The central mechanism
underlying hypertension: a review of the roles of sodium ions, epithelial sodium channels, therenin-angiotensin-aldosterone system, oxidative stress and endogenous digitalis in the brain.
Hypertension Res. 34, 1147-1160, 2011.
Yoshika, M., Komiyama, Y., and Takahashi, H. An ouabain-like factor is secreted fromimmortalized hypothalamic cells in an aldosterone-dependent manner. Neurochem. Int. 59,
104-108, 2011.
Yoshika, M., Komiyama, Y. and Takahashi, H. Isolation of marinobufotoxin from thesupernatant of cultured PC12 cells. Clin. Exp. Pharmacol. Physiol. 38, 334-337, 2011.

Spinal Cord Regeneration 脊髄再生医学

1.

Saito, F., Nakatani, T., Iwase, M., Maeda, Y., Murao, Y., Suzuki, Y., Fukushima, M., Ide, C.
Administration of cultured autologous bone marrow stromal cells into cerebtospinal fluid inspinal injury patients: a pilot study. Restor. Neurol. Neurosci. 30, 127-136, 2012.

Division of Cancer がん部門
Stem Cell Pathology 幹細胞病理学

1.
2.
3.
4.

Tanaka, T., Komai, Y., Tokuyama, Y., Yanai, H., Ohe, H., Okazaki, K. and Ueno, H.
Identification of stem cells that maintain and regenerate lingual keratinized epithelial cells. Nat.
Cell Biol. 15, 511-518, 2013.
Yanai, H., Tanaka, T. and Ueno, H. Multicolor lineage tracing methods and intestinal tumors. J.
Gastroenterology, 48, 423-433, 2013.
Zhang, H., Zheng, W., Shen, Y., Adhikari, D., Ueno, H. and Liu, K. Experimental evidence
showing that no mitotically active female germline progenitors exist in postnatal mouse ovaries.
Proc. Natl. Acad. Sci. U.S.A. 109, 12580-12585, 2012.
Rinkevich, Y., Lindau, P., Ueno, H., Longaker, M. and Weissman, I.L. Germ layer and lineage
restricted stem/progenitors regenerate the mouse digit tip. Nature 476, 409-413, 2011.

Viral oncology ウイルス腫瘍学

1.

2.

Nakamura, M., Kaneko, S., Ito, H., Jiang, S., Fujita, K., Wate, R., Nakano, S., Fujisawa, JI. andKusaka, H. Activation of transforming growth factor-β/Smad signaling reduces aggregate
formation of mislocalized TAR DNA-binding protein-43. Neurodegene. Dis. 11, 182-193,
2012.
Tanaka, M., Nitta, T., Sun, B., Fujisawa, J., Miwa, M. The route of primary HTLV-1 infection
regulates HTLV-1 distribution in reservoir organs of infected mice. Exp. Thr. Med. 2, 89-94,
2011.
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Stem Cell Biology 幹細胞生物学

1.
2.
3.

4.

Matsuoka, Y., Sasaki, Y., Nakatsuka, R., Takahashi, M., Iwaki, R., Uemura, Y. and Sonoda, Y.
Low level of c-kit expression marks deeply quiescent murine hematopoietic stem cells. StemCells 29, 1783-1791, 2011.
Kimura, T., Kohno, H., Matsuoka, Y., Nakatsuka, R., Sasaki, Y., Fukuhara, S. and Sonoda, Y.
CD16 antigen is a positive marker of peripheral blood-derived early endothelial progenitor cells.
Int. J. Hematol. 93, 123-125, 2011
Ishii, M., Matsuoka, Y., Sasaki, Y., Nakatsuka, R., Takahashi, M., Nakamoto, T., Yasuda, K.,
Matsui K, Asano, H., Uemura, Y., Tsuji, T., Fukuhara and Sonoda, Y. Development of ahigh-resolution purification method for precise functional characterization of primitive human
cord blood-derived CD34-negative SCID-repopulating cells. Exp. Hematol. 39, 203-213, 2011.
Kimura, T., Kohno, H., Matsuoka, Y., Murakami, M., Nakatsuka, R., Hase, M., Yasuda, K.,
Uemura, Y., Sasaki, Y., Fukuhara, S. and Sonoda Y. CXCL8 enhances the angiogenic activityof umbilical cord blood-derived outgrowth endothelial cells in vitro. Cell Biol. Int. 35, 201-208,2011.

Public Health 公衆衛生学

1.
2.
3.
4.

Yoshida, M., Hosomi, R., Fukunaga, K., Kanda, S. and Nishiyama, T. Tissue lanthanum
deposition and phosphorus balance in rats with long-term dietary administration of lanthanum
carbonate. Biomed. Res. Trace Elements 23, 40-44, 2012.
Tamada, Y., Kanda, S., Yoshidome, A., Hayashi, I., Miyake, M. and Nishiyama, T. Diagnosis ofactive tuberculosis using MPB64, a specific antigen of Mycobacterium bovis. Microbiol.
Immunol. 56, 740-747, 2012.
Ooka, H., Kanda, S., Okazaki, H., Suzuki, H., Mishima, K., Saito, I., Yagi, M., Tomoda, K. andNishiyama, T. Characterization of side population (SP) cells in murine cochlear nucleus. Acta
Otolaryngol. 132, 693-701, 2012.
Osumi, Y., Shibata, S. B., Kanda, S., Yagi, M., Ooka, H., Shimano, T., Mikiya A., Kawamoto,K., Kuriyama, H., Inoue, T., Nishiyama, T., Yamashita, T. and Tomoda, K. Downregulation of
N-methyl-D-aspartate receptor ζ1 subunit (GluN1) gene in inferior colliculus with aging. Brain
Res. 1454, 23-32, 2012.

Molecular Genetics 分子遺伝学部門

1.
2.
3.
4.

5.

6.

Ueda, Y., Katagiri, K., Tomiyama, T., Yasuda, K., Habiro, K., Katakai, T., Ikehara, S.,
Matsumoto, M. and Kinashi, T. Mst1 regulates integrin-dependent thymocyte trafficking andantigen-recognition in the thymus. Nat. Commun. 3, 1098, 2012.
Sekine, K., Kawauchi, T., Kubo, K., Honda, T., Herz, J., Hattori, M., Kinashi, T. and Nakajima,
K. Reelin controls neuronal migration and positioning by promoting neuronal adhesion to
extracellular matrix via the inside-out activation of integrin 51. Neuron 76, 353-369, 2012.
Fujii, Y., Shiota, M., Ohkawa, Y., Baba, A., Wanibuchi, H., Kinashi, T., Kurosaki, T. and Baba,
Y. Surf4 modulates STIM1-dependent calcium entry. Biochem. Biophys. Res. Commun. 422,
615-620, 2012.
Harada, Y., Tanaka, Y., Terasawa, M., Pieczyk, M., Habiro, K., Katakai, T., Hanawa-Suetsugu,
K., Kukimoto-Niino, M., Nishizaki, T., Shirouzu, M., Duan, X., Uruno, T., Nishikimi, A.,
Sanematsu, F., Yokoyama, S., Stein, J.V., Kinashi, T., and Fukui, Y. DOCK8 is a Cdc42
activator critical for interstitial dendritic cell migration during immune responses. Blood 119,4451-4461, 2012.
Hanawa-Suetsugu, K., Kukimoto-Niino, M., Mishima-Tsumagari, C., Akasaka, R., Ohsawa, N.,
Sekine, S., Ito, T., Tochio, N., Koshiba, S., Kigawa, T., Terada, T., Shirouzu, M., Nishikimi, A.,
Uruno, T., Katakai, T., Kinashi, T., Kohda, D., Tukui, Y. and Yokoyama, S. Structural basis formutual relief of the Rac guanine nucleotide exchange factor DOCK2 and its partner ELMO1
from their autoinhibited forms. Proc. Natl. Acad. Sci. USA. 109, 3305-3310, 2012.
Kinashi, T. Overview of integrin signaling in the immune system. Methods Mol. Biol. 757,
261-278, 2012.
22

Surgery 外科学

1.

2.

3.
4.
5.

6.
7.
8.

Yamao, J., Toyokawa, H., Kim, S., Yamaki, S., Satoi, S., Yanagimoto, H., Yamamoto, T.,
Hirooka, S., Matsui, Y. and Kwon, AH. Activation of alpha-smooth muscle actin-positive
myofibroblast-like cells after chemotherapy with gemcitabine in a rat orthotopic pancreaticcancer model. J. Hepatobiliary Pancreat. Sci. 20, 206-213, 2013.
Kaibori, M., Tanigawa, N., Kariya, S., Ikeda, H., Nakahashi, Y., Hirohara, J., Koreeda, C., Seki,
T., Sawada, S., Okazaki, K., and Kwon, AH. A prospective randomized controlled trial of
preoperative whole-liver chemolipiodolization for hepatocellular carcinoma. Dig. Dis. Sci. 57,
1404-1412, 2012.
Araki, Y., Kaibori, M., Matsumura, S., Kwon, AH. and Ito, S. Novel strategy for the control of
postoperative pain: long-lasting effect of an implanted analgesic hydrogel in a rat model of
postoperative pain. Anesth. Analg. 114, 1338-1345, 2012.
Kaibori, M., Ishizaki, M., Matsui, K. and Kwon, AH. Clinicopathologic characteristics ofpatients with non-B non-C hepatitis virus hepatocellular carcinoma after hepatectomy. Am. J.
Surg. 204, 300-307, 2012.
Inoue, K., Nakane, Y., Michiura, T., Yamada, M., Mukaide, H., Fukui, J., Miki, H., Ueyama,Y., Nakatake, R., Tokuhara, K., Iwamoto, S., Yanagimoto, H., Toyokawa, H., Satoi, S. andKwon, AH. Ultrasonic scalpel for gastric cancer surgery: a prospective randomized study. J.
Gastrointest. Surg. 16, 1840-1846, 2012.
Matsui, Y., Yamaki, S., Yamamoto, T., Isizaki, M., Matsui, K., Yanagimoto, H., Toyokawa, H.,
Kaibori, M., Satoi, S. and Kwon, AH. Absence of cystic duct leakage using locking clips in
1017 cases of laparoscopic cholecystectomy. Am. Surg. 78, 1228-1231, 2012.
Yokoigawa, N., Ogura, T. and Kwon, AH. Successful surgical control of peritoneal
dissemination of hepatocellular carcinoma. Case Rep. Gastroenterol. 6, 612-617, 2012.
Yamada, M., Yao, I., Hayasaka, T., Ushijima, M., Matsuura, M., Takada, H., Shikata, N., Setou,
M., Kwon, AH. and Ito, S. Identification of oligosaccharides from histopathological sections by
MALDI imaging mass spectrometry. Anal. Bioanal. Chem. 402, 1921-1930, 2012.

Dermatology 皮膚科学

1.

Mizuno, K., Suzuki, A., Kato, N., Isei, T., and Okamoto, H. CD8-positive granulomatous
mycosis fungoides mimicking generalized granuloma annulare. J. Dermatol. 39, 1068-1069,
2012.

Otolaryngology 耳鼻咽喉科学

1. Osumi, Y., Shibata, SB., Kanda, S., Yagi, M., Ooka, H., Shimano, T., Asako, M., Kawamoto,K., Kuriyama, H., Inoue, T., Nishiyama, T., Yamashita, T. and Tomoda, K. Downregulation of
N-methyl-D-aspartate receptor ζ1 subunit (GluN1) gene in inferior colliculus with aging. Brain
Res. 1454, 23-32, 2012.
2. Ooka, H., Kanda, S., Okazaki, H., Suzuki, H., Mishima, K., Saito, I., Yagi, M., Tomoda, K. andNishiyama, T. Characterization of side population (SP) cells in murine cochlear nucleus. Acta
Otolaryngol. 132, 693-701, 2012.
3. Shimano, T., Fyk-Kolodziej, B., Mirza, N., Asako, M., Tomoda, K., Bledsoe, S., Pan, ZH.,
Molitor, S. and Holt, AG. Assessment of the AAV- mediated expression of channelrhodopsin-2
and halorhodopsin in brainstem neurons mediating auditory signaling. Brain Res. 1511,
138-152, 2012.
Stem Cell Disorders 幹細胞異常症学1.
2.

Li, M., and Ikehara, S. Bone marrow stem cell as a potential treatment for diabetes. J. Diabetes
Res. 2013, 329596, 2013.
Li, M., and Ikehara, S. Bone-marrow-derived mesenchymal stem cells for organ repair. StemCells Int. 2013, 132642, 201323

Shi, M., Li, M., Cui, Y., Liu, L., Adachi, Y. and Ikehara S. CD4+ T cell-depleted lymphocyte
infusion impairs neither the recovery of recipient thymus nor the development of transplanted
thymus. J Immunol. 190, 2976-2983, 2013.
4. Shimo, T., Adachi, Y., Yamanouchi, S., Tsuji, S., Kimata, T., Umezawa, K., Okigaki, M,
Takaya, J., Ikehara, S. and Kaneko, K. A novel nuclear factor κB inhibitor,
dehydroxymethylepoxyquinomicin, ameliorates puromycin aminonucleoside-induced nephrosis
in mice. Am. J. Nephrol. 37, 302-309, 2013.
5. Zhang, Y., Hosaka, N., Cui, Y., Shi, M., Li, M., Li, Q. and Ikehara S. Effects of intra-bonemarrow-bone marrow transplantation plus adult thymus transplantation on survival of micebearing leukemia. Stem Cells Dev. 21, 1441-1448, 2012.
6. Shima, C., Adachi, Y., Minamino, K., Okigaki, M., Shi, M., Imai, Y., Yanai, S., Takahashi, K.
and Ikehara, S. Neuroprotective effects of granulocyte colony-stimulating factor on
ischemia-reperfusion injury of the retina. Ophthalmic Res. 48, 199-207, 2012.
7. Li, M., Vanella, L., Zhang, Y., Shi, M., Takaki, T., Shapiro, JI. and Ikehara, S. Stem celltransplantation increases antioxidant effects in diabetic mice. Int. J. Biol. Sci. 8, 1335-1344,
2012.
8. Zhang, Y., Hosaka, N., Cui, Y., Shi, M. and Ikehara, S. Effects of allogeneic hematopoietic
stem cell transplantation plus thymus transplantation on malignant tumors: Comparison
between fetal, newborn, and adult mice. Stem Cells Dev. 20, 599-607, 2011.
9. Oe, K., Kushida, T., Okamoto, N., Umeda, M., Nakamura, T., Ikehara, S. and Iida, H. New
strategies for anterior cruciate ligament partial rupture using bone marrow transplantation in
rats. Stem Cells Dev. 20, 671-679, 2011.
10. Shi, M., Lian, Z., Zhang, Y., Yanai, S., Shima, C., Imai, Y. and Ikehara, S. Combination of
intra-bone marrow-bone marrow transplantation and subcutaneous donor splenocyte injection
diminishes risk of GVHD and enhances survival rate. Stem Cells Dev. 20, 759-768, 2011.
11. Ikehara, S. Thymus transplantation for treatment of cancer: lessons from murine models. Expert
Rev. Clin. Immunol. 7, 205-211, 2011.
12. Mori, S., Fujita, S., Yamamoto, Y., Li, M., Fukuhara, S., Nomura, S. and Ikehara, S. Perfusion
method for bone marrow cell collection in poor mobilizer lymphoma patient. Int. J. Hematol.
93, 822-824, 2011.
13. Ikehara, S. A novel BMT technique for treatment of various currently intractable diseases. Best
Pract. Res. Clin. Haematol. 24, 477-483, 2011.
14. Hoshino, S., Kurishima, A., Inaba, M., Ando, Y., Fukui, T., Uchida, K., Nishio, A., Iwai, H.,
Yokoi, T., Ito, T., Ishii, S., Shimada, A., Li, M., Okazaki, K. and Ikehara, S. Amelioration of
2,4,6-trinitrobenzene sulfonic acid-induced colitis in mice by immunoregulatory dendritic cells.
J. Gastroenterol. 46, 1368-1381, 2011.
15. Zhang, Y., Hosaka, N., Cui, Y., Shi, M., Li, M., Li, Q. and Ikehara, S. Effects of intra-bonemarrow-bone marrow transplantation plus adult thymus transplantation on survival of micebearing leukemia. Stem Cells Dev. 21, 1441-1448, 2011.

3.

Division of Metabolic Disorders 代謝部門
Neuropharmacology 分子薬理学

1.

2.
3.

Noda, K., Dabovic, B., Takagi, K., Inoue, T., Horiguchi, M., Hirai, M., Fujikawa, Y., Akama,
T.O., Kusumoto, K., Zilberberg, L., Sakai, L.Y., Koki, K., Naitoh, M., von Melchner, H.,
Suzuki, S., Rifkin, D.B. and Nakamura, T. Latent TGFβ binding protein 4 promotes elastic
fiber assembly by interacting with fibulin-5. Proc. Natl. Acad. Sci. USA 110, 2852-2857, 2013.
Suzuki-Anekoji, S., Suzuki, A., Wu, S.W., Angata, K., Murai, K.K., Sugihara, K., Akama, T.O.,
Khoo, KH., Nakayama, J., Michiko N. and Fukuda, MN. In vivo regulation of steroid hormonesby the Chst10 sulfotransferase in mouse. J. Biol. Chem. 288, 5007-5016, 2013
Shibata, T.K., Matsumura, F. Wang, P., Yu, S., Choo, C.C., Khoo, K.H., Kitayama, K., Akama,
T.O., Sugihara, K., Kanayama, N., Kojima-Aikawa, K., Seeberger, P.H., Fukuda, M., Suzuki,
A., Aoki, D. and Fukuda, M.N. Identification of mono- and di-sulfated N-acetyllactosaminyl
oligosaccharide structures as epitopes specifically recognized by humanized monoclonal
antibody HMOCC-1 raised against ovarian cancer. J. Biol. Chem. 287, 6592-6602, 2012.
24

4.

5.
6.
7.
8.
9.

Horie, T., Baba, O., Kuwabara, Y., Chujo, Y., Watanabe, S., Kinoshita, M., Horiguchi, M.,
Nakamura, T., Chonabayashi, K., Hishizawa, M., Hasegawa, K., Kume, N., Yokode, M., Kita,
T., Kimura, T. and Ono, K. MicroRNA-33 deficiency reduces the progression of atherosclerotic
plaque in ApoE-/- mice. J. Am. Heart. Assoc. 1, e003376, 2012.
Otani, H., Yoshioka, K., Nishikawa, H., Inagaki, C. and Nakamura, T. Involvement of Protein
Kinase C and RhoA in Protease-Activated Receptor 1-Mediated F-Actin Reorganization andCell Growth in Rat Cardiomyocytes. J. Pharmacol. Sci. 115, 135-143, 2011.
Ohara, H., Akatsuka, S., Nagai, H., Liu, Y., Jiang, L., Okazaki, Y., Yamashita, Y., Nakamura,T. and Toyokuni, S. Stage-specific roles of fibulin-5 during oxidative stress-induced renal
carcinogenesis in rats. Free Radic. Res. 45, 211-220, 2011.
Tamura, N., Sugihara, K., Akama, T.O. and Fukuda, M.N. Trophinin-mediated cell adhesion
induces apoptosis of human endometrial epithelal cells through PKC-delta. Cell Cycle 10,135-143, 2011.
Parfitt, J.G., Pinali, C., Akama, T.O., Young, R.D., Nishida, K., Quantock, A.J. and Knupp, C.
Electron tomography reveals multiple self-association of chondroitin sulfate/dermatan sulfate
proteoglycans in Chst5-null mouse corneas. J. Struct. Biol.174, 536-541, 2011.
Hatakeyama, S., Sugihara, K., Shibata, K.T., Nakayama, J., Akama, T.O., Tamura, N., Wong,
S-M., Bobkov, A.A., Takano, Y., Ohyama, C., Fukuda, M., Fukuda, M.N. Targeted drugdelivery to tumor vasculature by a carbohydrate mimetic peptide. Proc. Natl. Acad. Sci. USA.
108, 19587-19592, 2011.

Cell Signaling 生体情報部門

1.
2.

3.
4.

5.

Takayama, G., Ohtani, M., Minowa, A., Matsuda, S. and Koyasu, S. Class I PI3K-mediated Akt
and ERK signals play a critical role in FceRI-induced degranulation in mast cells. Int. Immunol.
25, 215-220, 2013.
Powers, S.E., Mandal, M., Matsuda, S., Miletic, A.V., Cato, M.C., Tanaka, A., Rickert, R.C.,
Koyasu, S. and Clark, MR. Subnuclear cyclin D3 compartments and the coordinated regulationof proliferation and immunoglobulin variable gene repression. J. Exp. Med. 209, 2199-2213,
2012.
Ohtani, M., Hoshii, T., Fujii, H., Koyasu, S., Hirao, A. and Matsuda, S. mTORC1 in intestinal
CD11c+CD11b+ dendritic cells regulates intestinal homeostasis by promoting IL-10 production.
J. Immunol. 188, 4736-4740, 2012.
Kurebayashi, Y., Nagai, S., Ikejiri, A., Ohtani, M., Ichiyama, K., Baba, Y., Yamada, T., Egami,S., Hoshii, T., Hirao, A., Matsuda, S., and Koyasu, S. PI3K-Akt-mTORC1-S6K1/2 axis
controls Th17 differentiation by regulating Gfi-1 expression and nuclear translocation of RORg.
Cell Reports 1, 360-373, 2012.
Yokoyama, T., Matsuda, S., Takae, Y., Wada, N., Nishikawa, T., Amagai, M., and Koyasu, S.
Antigen-independent development of Foxp3+ regulatory T cells suppressing autoantibody
production in experimental pemphigus vulgaris. Int. Immunol. 23, 365-373, 2011.

Internal Medicine 1 内科学第一

1.
2.
3.
4.
5.

Ogata, M., Ito, T., Shimamoto, K., Nakanishi, T., Satsutani, N., Miyamoto, R. and Nomura, S.
Plasmacytoid dendritic cells have a cytokine-producing capacity to enhance ICOS
ligand-mediated IL-10 production during T-cell priming. Int. Immunol. 25, 171-182, 2013.
Fujita, S., Nakanishi, T., Yoshimura, H., Hotta, M., Nakamichi, N., Tamaki, T., Ishii, K., Ito, T.
and Nomura, S. TGFβ1 and sCTLA-4 levels are increased in eltrombopag-exposed patients with
ITP. Thromb. Res. 130, 415-419, 2012.
Nomura, S., Taniura, T., Shouzu, A., Omoto, S., Inami, N., Fujita, S., Tamaki, T., Yokoi, T.,
Shimizu, T., Ito, T. Effects of pitavastatin on plasminogen activator inhibitor-1 in
hyperlipidemic patients. Int. J. Gen. Med. 5, 535-540, 2012.
Ito, T. and Arima, K. Cellular and molecular mechanisms of TSLP function in human allergic
disorders--TSLP programs the “Th2 code” in dendritic cells--. Allergol. Int. 61, 35-43, 2012.
Ito, T. Plasmacytoid dendritic cells and type I interferon as therapeutic cellular and moleculartargets of autoimmune diseases. Cytometry Res. 22, 37-46, 2012.
25

6.

Nomura, S., Fujita, S., Nakanishi, T., Yokoi, T., Shimamoto, K., Miyamoto, R., and Ito, T.
Platelet-derived microparticles cause CD154-dependent activation of dendritic cells. Platelets
23, 81-82, 2011.
7. Lande, R., Ganguly, D., Facchinetti, V., Frasca, L., Conrad, C., Gregorio, J., Meller, S.,
Chamilos, G., Sebasigari, R., Riccieri, V., Bassett, R., Amuro, H., Fukuhara, S., Ito, T., Liu, YJ.
and Gilliet, M. Neutrophils Activate Plasmacytoid Dendritic Cells by Releasing
Self-DNA-Peptide Complexes in Systemic Lupus Erythematosus. Sci. Transl. Med. 3, 73ra19,
2011.
8. Hoshino, S., Kurishima, A., Inaba, M., Ando, Y., Fukui, T., Uchida, K., Nishio, A., Iwai, H.,
Yokoi, T., Ito, T., Hasegawa-Ishii, S., Shimada, A., Li, M., Okazaki, K. and Ikehara, S.
Amelioration of 2,4,6-trinitrobenzene sulfonic acid-induced colitis in mice byimmunoregulatory dendritic cells. J. Gastroenterol. 46, 1368-1381, 2011.
9. Katashiba, Y., Miyamoto, R., Hyo, A., Shimamoto, K., Murakami, N., Ogata, M., Amakawa, R.,
Inaba, M., Nomura, S., Fukuhara, S. and Ito, T. Interferon-α and interleukin-12 are induced,
respectively, by double-stranded DNA and single-stranded RNA in human myeloid dendritic
cells. Immunology 132, 165-173, 2011.
10. Nomura, S., Fujita, S., Ozasa, R., Nakanishi, T., Miyaji, M., Mori, S., Ito, T. and Ishii, K. The
correlation between platelet activation markers and HMGB1 in patients with disseminatedintravascular coagulation and hematologic malignancy. Platelets 22, 396-397, 2011.
11. Nomura, S., Ozasa, R., Nakanishi, T., Fujita, S., Miyaji, M., Mori, S., Yokoi, T., Ito, T. andIshii, K. Can recombinant thrombomodulin play a preventive role for veno-occlusive disease
after haematopoietic stem cell transplantation? Thromb. Haemost. 105, 1118-1120, 2011.
12. Nomura, S., Nakanishi, T., Ozasa, R., Fujita, S., Miyaji, M., Mori, S., Yokoi, T., Ishii, K. andIto, T. Dynamic role of cell-derived microparticles in hematopoietic stem cell transplantation.
Curr. Trends Immunol. 12, 21-27, 2011.
Internal Medicine 2 内科学第二1.

Haiden, M., Miyasaka, Y., Kimura, Y., Tsujimoto, S., Maeba, H., Suwa, Y., Iwasaka, T.,
Shiojima, I. Effect of eicosapentaenoic acid on regional arterial stiffness: Assessment by tissue
Doppler imaging. World J. Cardiol. 4, 256-259, 2012.

Inernal Medicine 3 内科学第三

1.

2.

3.

4.
5.
6.
7.

Yamaguchi, T., Matsuzaki, K., Inokuchi, R., Kawamura, R., Yoshida, K., Murata, M., Fujisawa,J., Fukushima, N., Sata, M., Kage, M, Nakashima, O., Tamori, A., Kawada, N., Tsuneyama, K.,
Dooley, S., Seki, T. and Okazaki, K. Phosphorylated Smad2 and Smad3 signaling: Shifting
between tumor suppression and fibro-carcinogenesis in chronic hepatitis C. Hepatol. Res. inpress, 2013.
Miyoshi, H., Kusuda, T., Kurishima, A., Fukui, Y., Sumimoto, K., Satoi, S., Ohe, C., Uemura,Y., Kwon, AH. and Okazaki, K. Autoimmune pancreatitis with histologically provenlymphoplasmacytic sclerosing pancreatitis with granulocytic epithelial lesions. Intern. Med. 51,
733-737, 2012.
Uchida, K., Kusuda, T., Koyabu, M., Miyoshi, H., Fukata, N., Sumimoto, K., Fukui, Y.,
Sakaguchi, Y., Ikeura, T., Shimatani, M., Fukui, T., Matsushita, M., Takaoka, M., Nishio, A.
and Okazaki, K. Regulatory T cells in type 1 autoimmune pancreatitis. Int. J. Rheumatol. 2012,
795026, 2012.
Okazaki, K. and Umehara, H. Are Classification Criteria for IgG4-RD Now Possible? The
concept of IgG4-related disease and proposal of comprehensive diagnostic criteria in Japan. Int.
J. Rheumatol. 2012, 357071, 2012.
Yamashina, M., Nishio, A., Nakayama, S., Okazaki, T., Uchida, K., Fukui, T. and Okazaki, K.
Comparative study on experimental autoimmune pancreatitis and its extrapancreatic
involvement in mice. Pancreas 41, 1255-1262, 2012.
Umehara, H., Seki, T., Inokuchi, R., Tamai, T., Kawamura, R., Asayama, T., Ikeda, K. andOkazaki, K. Microwave coagulation using a perfusion microwave electrode: Preliminary
experimental study using ex vivo and in vivo liver. Exp. Ther. Med. 3, 214-220, 2012.
Fukui, Y., Uchida, K., Sumimoto, K., Kusuda, T., Miyoshi, H., Koyabu, M., Ikeura, T.,
Sakaguchi, Y., Shimatani, M., Fukui, T., Matsushita, M., Takaoka, M., Nishio, A., Shikata, N.,26

Sakaida, N., Uemura, Y., Satoi, S., Kwon, AH. and Okazaki, K. The similarity of Type 1
autoimmune pancreatitis to pancreatic ductal adenocarcinoma with significant IgG4-positive
plasma cell infiltration. J. Gastroenterol. 48, 751-761, 2012.
8. Matsushita, M., Fukata, N., Omiya, M. and Okazaki, K. Absence of large ulcers indicate thatviral therapy is not necessary in cytomegalovirus infection in ulcerative colitis. J. Clin.
Gastroenterol. 47, 90-91, 2013.
9. Watanabe, T., Yamashita, K., Fujikawa, S., Sakurai, T., Kudo, M., Shiokawa, M., Kodama, Y.,
Uchida, K., Okazaki, K. and Chiba, T. Involvement of activation of toll-like receptors andnucleotide-binding oligomerization domain-like receptors in enhanced IgG4 responses in
autoimmune pancreatitis. Arthritis Rheum. 64, 914-924, 2012.
10. Nishio, A., Asada, M., Uchida, K., Fukui, T., Chiba, T. and Okazaki, K. The role of innate
immunity in the pathogenesis of experimental autoimmune pancreatitis in mice. Pancreas 40,
95-102, 2011.
11. Okazaki, K., Uchida, K, Miyoshi, H., Ikeura, T., Takaoka, M. and Nishio, A. Recent conceptsof autoimmune pancreatitis and IgG4-Related disease. Clin. Rev. Allergy Immunol. 41, 126-138,
2011.
12. Fukui, T., Kishimoto, M., Nakajima, A., Yamashina, M., Nakayama, S., Kusuda, T., Sakaguchi,
Y., Yoshida, K., Uchida, K., Nishio, A., Matsuzaki, K. and Okazaki, K. The specific linker
phosphorylation of Smad2/3 indicates epithelial stem cells in stomach; particularly increasing
in mucosae of Helicobacter-associated gastritis. J. Gastroenterol. 46, 456-468, 2011.
13. Okazaki, K., Uchida, K., Koyabu, M., Miyoshi, H. and Takaoka, M. Recent advances in theconcept and diagnosis of autoimmune pancreatitis and IgG4-related disease. J. Gastroenterol.
46, 277-288, 2011.
14. Hoshino, S., Kurishima, A., Inaba, M., Ando, Y., Fukui, T., Uchida, K., Nishio, A., Iwai, H.,
Yokoi, T., Ito, T., Hasegawa-Ishii, S., Shimada A, Li M, Okazaki K. and Ikehara S.
Amelioration of 2,4,6-trinitrobenzene sulfonic acid-induced colitis in mice byimmunoregulatory dendritic cells. J. Gastroenterol. 46, 1368-1381, 2011.
15. Kusuda, T., Uchida, K., Miyoshi, H., Koyabu, M., Satoi, S., Takaoka, M., Shikata, N., Uemura,Y. and Okazaki, K. Involvement of inducible costimulator- and interleukin 10-positive
regulatory T cells in the development of IgG4-related autoimmune pancreatitis. Pancreas 40,
1120-1130, 2011.
16. Nakase, H., Tamaki, H, Matsuura, M., Chiba, T. and Okazaki, K. Involvement of
mycobacterium avium subspecies paratuberculosis in TNF-α production from macrophage:possible link between MAP and immune response in Crohn's disease. Inflamm. Bowel Dis. 17,
E140-142, 2011.
17. Ikeura, T., Takaoka, M., Uchida, K., Shimatani, M., Miyoshi, H., Kusuda, T., Kurishima, A.,
Fukui, Y., Sumimoto, K., Satoi, S., Ohe, C., Uemura, Y., Kwon, AH. and Okazaki, K.
Autoimmune pancreatitis with histologically proven lymphoplasmacytic sclerosing pancreatitis
with granulocytic epithelial lesions. Intern. Med. 51, 733-737, 2012.
18. Nakajima, A., Fukui, T., Takahashi, Y., Kishimoto, M., Yamashina, M., Nakayama, S.,
Sakaguchi, Y., Yoshida, K., Uchida, K., Nishio, A., Yodoi, J. and Okazaki, K. Attenuation ofindomethacin-induced gastric mucosal injury by prophylactic administration of sakeyeast-derived thioredoxin. J. Gastroenterol. 47, 978-987, 2012.
Plastic and Reconstructive Surgery 形成外科学

1.
2.

3.
4.

Kakudo, N., Morimoto, N., Kushida, S., Ogawa, T. and Kusumoto, K. Platelet-rich plasma
releasate promotes angiogenesis in vitro and in vivo. Med. Mol. Morphol. in press, 2013.
Kakudo, N., Kushida, S., Suzuki, K., Ogura, T., Notodihardjo, P.V., Hara, T. and Kusumoto, K.
Effects of transforming growth factor-beta1 on cell motility, collagen gel contraction,
myofibroblastic differentiation, and extracellular matrix expression of human adipose-derived
stem cell. Human Cell 25, 87-95, 2012.
Kushida, S., Kakudo, N., Suzuki, K. and Kusumoto, K. Effects of platelet-rich plasma on
proliferation and myofibroblastic differentiation in human dermal fibroblasts. Ann. Plast. Surg.
71, 219-224, 2013.
Notodiharjo, F.Z., Kakudo, N., Kushida, S., Suzuki, K. and Kusumoto, K. Bone regeneration
with BMP-2 and hydroxyapatite in critical-size calvarial defects in rats. J. Craniomaxillofac.
Surg. 40, 287-291, 2012.
27

5.
6.

Kakudo, N., Kushida, S., Minakata, T., Suzuki, K. and Kusumoto, K. Platelet-rich plasma
promotes epithelialization and angiogenesis in a splitthickness skin graft donor site. Med. Mol.
Morphol. 44, 233-236, 2011.
Kakudo, N., Kushida, S., Suzuki, K., Matsumoto, N., Kusumoto K. The effect of C3 transferaseon human adipose-derived stem cells. Human Cell 24, 165-169, 2011.

Ophthalmology 眼科学

1.
2.
3.
4.
5.

Kimura, M., Ogata, N., Shima, C. and Takahashi, K. Choroidal melanoma with massive
extraocular extensions through sclera. Clin. Ophthalmol. 6, 2081-2084, 2012.
Yoshikawa, T., Nishimura, T., Minamino, K. and Takahashi, K. A long-term follow-up of
peripapillary retinoschisis with optic disc hypoplasia. Int. Ophthalmol. 33, 425-428, 2012.
Matsuoka, M., Ogata, N., Matsuyama, K., Yoshikawa, T. and Takahashi, K. Intraocularpressure in Japanese diabetic patients. Clin. Ophthalmol. 6, 1005-1009, 2012.
Tateno, H., Taomoto, M., Jo, N., Takahashi, K. and Uemura, Y. Clinical and histopathologicalfeatures of a suspected case of fish-eye disease. Jpn. J. Ophthalmol. 56, 453-457, 2012.
Shima, C., Adachi, Y., Minamino, K., Okigaki, M., Shi, M., Imai, Y., Yanai, S., Takahashi, K.
and Ikehara, S. Neuroprotective effects of granulocyte colony-stimulating factor on
ischemia-reperfusion injury of the retina. Ophthalmic. Res. 48, 199-207, 2012.

Radiology 放射線科学

1.

Matsumura, Y., Shinjo, M., Matsui, T., Ichimura, K., Song, J. and Kihara H. Structural study ofhNck2 SH3 domain protein in solution by circular dichroism and X-ray solution scattering.
Biophys. Chem. 175-176, 39-46. 2013.

2.

Kariya, S., Komemushi, A., Nakatani, M., Yoshida, R., Sawada, S. and Tanigawa, N. CO2
microbubble contrast enhancement in x-ray angiography. Clin. Radiol. 68, 346-351, 2013.

3.

Takemoto, K., Ikegaya, H., Yaji, T., Ichise, S., Ohta, T. and Kihara, H. Infrared
Micro-Spectroscopy Observation of plantplankton with in Lake Biwa. Memoirs of the SR
Center, Ritsumeikan University, 14, 173-17. 2012.

4.

Nakatani, M., Tanigawa, N., Kariya, S., Komemushi, A., Yagi, R. and Sawada, S. Analysis offactors influencing accuracy and complications in CT-guided lung biopsy. Minim. Invasive Ther.
Allied Technol. 21, 415-422, 2012.

28

IV.

セミナー、シンポジウム、できごと

2011 年 9 月
滝井キャンパス 2 号館北棟 4 階生化学室 2 と北棟 3 階 光学顕微鏡室 3 に近赤外蛍光イメー
ジャーOdyssey system と HS オールインワン蛍光顕微鏡がそれぞれ導入されました。
2011 年 12 月 9 日
滝井キャンパス南館臨床講堂にて高次機能制御系・ブレインメディカルサイエンス系
学院研究中間発表会中間発表会が開催されました。

大2011 年 12 月 16 日
滝井キャンパスセミナールーム A にて Assist. Prof. Mino Mari Kenduson (Dept. of Pathology,
Massachusetts General Hospital) による「Pathological aspects of Intrapancretic papillary mucosal
neoplasia (IPMN) in USA」（岡崎和一教授司会）と題して大学院講座が開催されました。
2012 年 3 月 22 日
附属枚方病院 13 階講堂にて慶應義塾大学医学部生理学教室 岡野 栄之教授をお招きし、「iPS 細胞技術を用いた神経再生・疾患・創薬研究」（今村洋二枚方病院長司会、淀川リバ
ーサイズメディカルサポートプログラム・病態分子イメージングセンター共催）と題する
病態分子イメージングセンターキックオフ特別講演会が開催されました。ビデオ収録（ビデオはイントラネットのサイトで「病態分子」と検索すると見られます。）
2012 年 3 月 27、28 日
滝井キャンパス２号館北棟 2 階 電子顕微鏡室に質量顕微鏡が設置され、利用希望者に講習会が開催されました。4 月 25、27 日にも講習会が開催されました。
2012 年 4 月 27 日
滝井キャンパス 1 号館大会議室にて Learning & Development, Alzheimer's Australia Victoria,
General Manager, David Sykes 博士による「Care for People with Dementia」、Dance-Movement
Therapy Association of Australia, Heather Hill 教授による「Dementia and Dance Therapy」（三宅眞理講師司会）と題する大学院講義が開催されました。
2012 年 5 月 1 日
附属枚方病院 13 階講堂にて山本雅先生 (沖縄科学技術大学院大学教授、前東京大学医科学研究所所長)、Min Zhuo 教授（University of Toronto, Canada）、John A. Hanover 博士（Lab Chief,
NIDDKD, NIH, USA）を外部評価委員として、病態分子イメージングセンター第 1 回 Annual
meeting を開催しました。
2012 年 5 月 11 日
滝井キャンパス南館臨床講堂にて大学院総合講義、綜合研究施設利用講習会で矢尾育子講師（医化学）が「質量顕微鏡の応用例」を発表しました。
2012 年 5 月 18 日
滝井キャンパス南館臨床講堂にて大学院総合講義、ラジオアイソトープ実験施設利用講習会で国立循環器病研究センター画像診断医学部 飯田秀博部長による「核医学分子イメー
ジングにおける定量化について」（宇都宮啓太講師司会）と題した講演が行われました。
2012 年 6 月 18 日
滝井キャンパス精神医療総合センターにて京都ノートルダム女子大学心理学部心理学科
藤川 洋子教授による「英国、スウェーデンの触法発達障害者に対する治療と支援」（木下利彦教授司会）と題した大学院講座が開催されました。

29

2012 年 6 月 22 日
滝井キャンパスセミナールーム B にて Emory 大学医学部小児科 近藤 直幸博士による「膜融合を介した HIV の侵入と細胞融合のキネティクス宿主因子によるその調整」（木梨達雄教授司会）と題した大学院講座が開催されました。
2012 年 8 月 6 日
滝井キャンパスセミナーA にて British Columbia 大学脳研究センター精神医学講座 高橋 秀
人研究員による「新規シナプス接着分子の探索と機能解析」（岡田誠剛講師司会）と題した大学院講座が開催されました。
2012 年 10 月 18 日
滝井キャンパスセミナールーム S にて川崎医科大学微生物学教室 齊藤 峰輝教授による「HTLV-1 等の慢性ウイルス感染症による中枢神経障害の発症機序」（藤澤順一教授司会）と題した大学院講座が開催されました。
2012 年 10 月 30 日
滝井キャンパスセミナーA にて Massachusetts General Hospital 病理学講座 Mino Mari
Kenduson 博士による「Ductal neoplasms of the pancreas：with an emphasis on molecularalterations」（岡崎和一教授司会）と題した大学院講座が開催されました。
2012 年 12 月 17 日
枚方病院第 3 会議室にて昭和大学医学部外科学講座小児外科部門 土岐 彰教授の「小児鼠径ヘルニアの超音波診断」（濱田吉則准教授司会）と題した大学院講座が開催されました。
2012 年 12 月 20 日
病態分子イメージングセンターの RI イメージングルームを含む RI 実験施設の使用許可証が交付されました。
2013 年 1 月 18 日
滝井キャンパスセミナールーム A にて産業技術総合研究所 バイオメディカル研究部門
近江谷 克裕研究部門長による「医療イノベーションを目指した光イメージングの最前線」（伊藤誠二教授司会）と題した大学院講座が開催されました。
2013 年 2 月～3 月
枚方学舎竣工により、病態分子イメージングセンターを滝井キャンパスから枚方キャンパスに移転しました。
2013 年 3 月 21 日
枚方病院第 1 会議室にて長崎大学大学院医歯薬学総合研究科 感染免疫学講座 先進感染制御学分野 中村 龍文准教授の「HTLV-1 関連脊髄症（HAM）に対する新規治療戦略」（藤澤順一教授司会）と金沢医科大学医学部 微生物学講座 大原 義朗教授の「新しいウイル
ス感染症～ヒトカルジオウイルスについて～」（藤澤順一教授司会）と題した大学院講座が開催されました。
2013 年 3 月 22 日
枚方病院 13 階合同カンファレンスルームにて理化学研究所 発生・再生科学総合研究セン
ター 器官発生研究グループ 笹井 芳樹グループディレクターをお招きし、「三次元培養
での幹細胞から機能的な器官の自己形成」（病態分子イメージングセンター主催、伊藤誠二
センター長司会）と題した特別講演会が開催されました。ビデオ収録（ビデオはイントラ
ネットのサイトで「病態分子」と検索すると見られます。
）
2013 年 4 月 12 日
枚方学舎 5 階病態分子イメージングセンター
されました。
30

質量顕微鏡室にて質量顕微鏡講習会が開催

2013 年 4 月 12 日
枚方学舎 4 階会議室 D にて産業医科大学名誉教授・臨床研究推進センター 笹栗 靖之セン
ター長（前第 2 病理学講座教授）の「臨床、基礎医学の研究における病理学的アプローチの有用性について」（伊藤誠二教授司会）と題した大学院講座が開催されました。
2013 年 4 月 15 日
枚方学舎 1 階第 1 講義室にて千葉県こども病院小児外科 岩井 潤部長の「総排泄腔遺残症に対する Total urogenital mobilization（TUM）」（濱田吉則准教授司会）と題した大学院講座が開催されました。
2013 年 4 月 17 日
枚方学舎 4 階カンファレンスルーム D にて Baylor 大学医学部 Jan and Dan Duncan 神経研究所 分 子 ヒ ト 遺 伝 学 部 門 山 本 慎 也 室 長 の 「 Identification of novel genes in neuronaldevelopment, function and maintenance through forward genetic approaches in Drosophila」（矢尾育子准教授司会）と題した大学院講座が開催されました。
2013 年 4 月 17 日
枚方学舎 2 階第 4 講義室にて Weizmann 科学研究所免疫学講座 Tsvee Lapidot 教授による「Regulation of normal and leukemic human stem cells」（薗田精昭教授司会）と題した大学院講座が開催されました。
2013 年 4 月 26 日
枚方学舎 1 階加多乃講堂にて東京都立駒込病院 神澤 輝実博士をお招きし、「自己免疫性膵炎」（岡崎和一教授司会）と題した大学院講座が開催されました。
2013 年 5 月 24 日
枚方学舎 1 階加多乃講堂にてラジオアイソトープ実験施設利用講習会で国立長寿医療研究センター脳機能画像診断開発部 鈴木 正昭特任研究員（前理科学研究所分子イメージング
科学研究センター副センター長）による「高速カップリング反応による短寿命 PET 分子プ
ローブの革新的合成法と生物学的活用」（伊藤誠二教授司会）と題した講演が行われました。
2013 年 6 月 15 日
枚方学舎 4 階中会議室にて山本 雅教授（沖縄科学技術大学院大学、前東京大学医科学研究所所長）、Min Zhuo 教授（University of Toronto, Canada）、John A. Hanover 博士（Lab Chief,
NIDDKD, NIH, USA）を外部評価委員として、病態分子イメージングセンター第 2 回 Annual
meeting が開催されました。
2013 年 6 月 25 日
枚方学舎 4 階カンファレンスルーム D にて Laboratory of Sensorimotor Research, National Eye
Institute, NIH, Ethan Bromberg-Martin 博士による「Neural circuits for information seeking」（中村加枝教授司会）と題した講演が行われました。
2013 年 8 月 5 日
滝井学舎南館臨床講堂にて森島 陽介先生による「脳領域間の機能的結合：基礎から治療応用を目指して」（木下利彦教授司会）と題した大学院講座が開催されました。

31

IV. Seminars, symposia, and events of MICD
September 2011
Infrared imaging system Odyssey and HS all-in-one fluorescence microscope were installed at
Biochemistry room 2 and Optical microscope room 3, respectively, of the north block of Building 2
in Takii campus.
December 9, 2011
Interim Presentation of Graduate School by third-year graduate students of Integrative Biosystem
of Medical Science course and Medical Neuroscience of Advanced Medicine course at ClinicalAuditorium of Takii campus.
December 16, 2011
Graduate lecture “Pathological aspects of intrapancretic papillary mucosal neoplasia (IPMN) in
USA” by Assistant Prof. Mino Mari Kenduson (Dept. of Pathology, Massachusetts General Hospital,
USA) was held at Seminar room A of Bld. 1 in Takii campus (sponsored by Prof. Kazuichi Okazaki).
March 22, 2012
Kick-off lecture “Neural regeneration and application of neural disorders by iPS cell technology”
by Prof. Hideyuki Okano (Dept. of Physiology, Kaio University) was held at 13th-floor auditorium
in Harakata hospital (sponsored by Molecular Imaging Center of Diseases).
March 27, 28, 2012
Imaging mass spectrometry was installed at Electron microscope room on the 2nd floor of thenorth block of Building 2 in Takii campus. A technical institute of imaging mass spectrometry tousers was held. The second institute was held on April 25 and 27.
April 27, 2012
Graduate lecture “Care for people with dementia” by Dr. David Sykes (Alzheimer's Australia
Victory) and Graduate lecture: Dementia and dance therapy by Dr. Heather Hill (Dance-Movement
Therapy Association of Australia) were held at 5th Grand Conference Room of Building 1 in Takii
campus (sponsored by Dr. Mari Miyake).
May 1, 2012
The 1st annual meeting of Molecular Imaging Center of Diseases was held at 13th-floor auditorium
in Harakata hospital in the presence of Drs. Tadashi Yamamoto, Min Zhuo, and John Hanover,advisory board members of Molecular Imaging Center of Diseases.
May 11, 2012
A technical institute of “Application of imaging mass spectrometry by Dr. Ikuko Yao (Medical
Chemistry) as a topic of Graduate lecture was held at Clinical Auditorium in Takii campus(organized by General Research Center).
May 18, 2012
Graduate lecture “A lecture concerning imagingtechnics and application” by Dr. Hidehiro Iida
(Dept. of Bio-medical Imaging, National Cerebral and Cardiovascular Center Research Institute)
was held at Clinical Auditorium in Takii campus (organized by Radioisotope Center).
June 18, 2012
Graduate lecture “Treatment and support to patients with disruptive behavior disorders in United
Kingdom and Sweden” by Prof. Yoko Fujikawa (Dept. of Psychology, Kyoto Notre Dame
University) was held at Psychiatric Center in Takii campus (sponsored by Prof. Toshihiko
Kinoshita).

32

June 22, 2012
Graduate lecture “Invasion of HIV by membrane fusion, kinetics of cell fusion and its regulation
by host factors” by Dr. Naoyuki Kondo (Dept. of Pediatrics, Emory University School of Medicine)
was held at Seminar Room B in Takii campus (sponsored by Prof. Tatsuo Kinashi).
August 6, 2012
Graduate lecture ”Search and functional analysis of novel glue molecules at synapse” by Dr.
Hideto Takahashi (Brain Research Centre and Dept. of Psychiatry, University of British Columbia)
was held at Seminar Room A in Takii campus (sponsored by Associate Prof. Masanobu Okada).
October 18, 2012
Graduate lecture “Pathogenesis of disturbance in central nervous system by chronic viral infection
including HTLV-1” by Prof. Mineki Saito (Dept. of Microbiology, Kawasaki Medical School) was
held at Seminar Room S in Takii campus (sponsored by Prof. Junichi Fujisawa).
October 30, 2012
Graduate Seminar “Ductal neoplasms of the pancreas：with an emphasis on molecular alterations"by Assist. Prof. Mino Mari Kenduson (Dept. of Pathology, Massachusetts General Hospital) was
held at Seminar Room A in Takii campus (sponsored by Prof. Kazuichi Okazaki).
December 17, 2012
Graduate lecture “Ultrasonic diagnosis of inguinal hernia in children” by Prof. Akira Toki (Div. of
Pediatric Surgery, Showa University) was held at Conference room 3 in Hirakata Hospital(sponsored by Associate Prof. Yoshinori Hamada).
December 20, 2012
Usage permission of RI imaging room in Molecular Imaging Center of Diseases was issued by the
Ministry of Education, Culture, Sports, Science and Technology.
January 18, 2013
Graduate lecture “Front of photoimaging directed medical service innovation” by Dr. Yoshihiro
Ohmiya (Director, Biomedical Research Institute, AIST) was held at Seminar room A in Takii
campus (sponsored by Prof. Seiji Ito).
February –March, 2013
Research facilities in Molecular Imaging Center of Diseases and Laboratories were moved from
Takii campus to Hirakata campus.
March 21, 2013
Graduate lectures “New strategy of treatment to HTLV-I-associated myelopathy (HAM)” by
Associate Prof. Tatsufumi Nakamura (Div. of Molecular and Clinical Microbiology, Nagasaki
University Graduate School of Biomedical Sciences) and “New viral infectious disease –humancardiovirus” by Prof. Yoshiro Ohara (Dept. of Microbiology, Kanazawa Medical University) were
held at Conference room D in Hirakata campus (sponsored by Prof. Jun-ichi Fujisawa).
March 22, 2013
Special lecture sponsored by Molecular Imaging Center of Diseases: ”Self-formation of functionalorgan from stem cells by three-dimensional culture” by Dr. Yoshiki Sasai (Director, Organogenesis
and Neurogenesis Group, Center for Developmental Biology, RIKEN) was held at 13th-floor Joint
Conference room in Hirakata campus.
April 12, 2013
A technical institute of imaging mass spectrometry to users was held at Electron microscope room
in Molecular Imaging Center of Diseases.
Graduate lecture “Utility of pathological approach in clinical and basic research” by Dr. Yasuyuki
Sasaguri (Emeritus Professor, Director of Clinical trial research center, University of Occupational
and Environmental Health) was held at Conference room D in Hirakata campus (sponsored by Prof.
Seiji Ito).
33

April 15, 2013
Graduate lecture “Total urogenital mobilization toward persistent cloaca” by Dr. Jun Iwai (Chiefof Pediatric Surgery, Chiba Children’s Hospital) was held at Lecture room 1 in Hirakata campus(sponsored by Associate Prof. Yoshinori Hamada).
.
April 17, 2013
Graduate lecture “Identification of novel genes in neuronal development, function andmaintenance through forward genetic approaches in Drosophila” by Dr. Shinya Yamamoto (NRI
Fellow and Lab Head, Dept. of Molecular and Human Genetics, Jan and Dan Duncan NeurologicalResearch Institute, Baylor College of Medicine) was held at Conference room D in Hirakata campus(sponsored by Associate Prof. Ikuko Yao).
Graduate lecture “Regulation of normal and leukemic human stem cells” by Prof. Tsvee Lapidot(Dept. of Immunology, Weizmann, Institute of Science, Israel) was held at Lecture room 4 in
Hirakata campus (sponsored by Prof. Yoshiaki Sonoda).
April 26, 2013
Graduate lecture “Autoimmune pancreatitis” by Dr. Terumi Kamisawa (Director of Internal
medicine, Dept. of Internal Medicine, Tokyo Metropolitan Komagome Hospital) was held at Katano
Lecture Hall in Hirakata campus (sponsored by Prof. Kazuichi Okazaki).
May 24, 2013
Graduate lecture “Revolutionary synthetic method of short-life PET probe by high-speed coupling
reaction and its medical application” by Dr. Masaaki Suzuki (Dept. of Clinical and Experimental
Neuroimaging, National Center for Geriatrics and Gerontology) was held at Katano Lecture Hall in
Hirakata campus (organized by Radioisotope Center).
June 15, 2013
The 2nd annual meeting of Molecular Imaging Center of Diseases was held at 4th-floor Middle
Conference Room in Harakata campus in the presence of Drs. Tadashi Yamamoto, Min Zhuo, andJohn Hanover, advisory board members of Molecular Imaging Center of Diseases.
June 25, 2013
Graduate lecture “Neural circuits for information seeking” by Dr. Ethan Bromberg-Martin(Laboratory of Sensorimotor Research, National Eye Institute, NIH) was held at Conference room D
in Hirakata campus (sponsored by Prof. Kae Nakamura).
August 5, 2013
Graduate lecture “Functional connection among brain regions: toward therapy from basic
research” by Dr. Yosuke Morishima was held at Clinical Auditorium in Takii campus (sponsored by
Prof. Toshihiko Kinoshita).

34

The 1st Annual Meeting of Molecular Imaging Center of Diseases
on May 1, 2012 at 13th-floor Auditorium, Hirakata Hospital

Program
1:00-1:10
1:10-1:30

1:30-2:30

Opening remarks
Introduction of advisory board members
Outline and prospect of Molecular Imaging Center of Diseases
Seiji Ito
Session 1: Division of Metabolic Disorders
Advisory Board: Dr. John A. Hanover, USA
Chair: Tomoyuki Nakamura, Kazuichi Okazaki

1:30-1:45 Tomoyuki Nakamura (Molecular Pharmacology)
Development and regeneration of elastic fiber
1:45-2:00 Hiroyasu Tsukaguchi （Internal Medicine 2）
Genetic approaches to identify new targets of molecular imaging for kidney andneurodevelopmental disorders
2:00-2:15 Toshiro Fukui (Internal Medicine 3)
The specific linker phosphorylation of Smad2/3 indicates epithelial stem cells incolon; particularly increasing in the regeneration phase of murine model of dextran
sulfate sodium-induced colitis
2:15-2:30 Tomoki Ito (Internal Medicine 1)
Dendritic cells as therapeutic targets of autoimmune diseases.

2:30-3:50

Session 2: Division of Neuroscience
Advisory Board: Prof. Min Zhuo, Canada
Chair: Tetsuo Sugimoto, Hirofumi Kusaka

2:30-2:42 Masayoshi Okada (Cellular and Molecular Physiology)
The degradation of the inwardly rectifying potassium channel, Kir2.1, depends on thecurrent level
2:42-2:54 Taketoshi Wakabayashi (Anatomy and Cell Science)
Cell type specific expression of lamin subtypes in rat retinal neurons
2:54-3:06 Hakuo Takahashi (Clinical Sciences and Laboratory Medicine)
Analysis of the brain digitalis-like substance underlying hypertension
3:25-3:37 Satoshi Kaneko (Neurology)
Analysis of intracytoplasmic inclusions of neurodegenerative diseases by imaging
mass spectrometry
3:37-3:50 Masahito Shimojo (Molecular and Functional Biology)
The challenges of neurodegenerative disease modeling using patient-specific iPSCs
35

3:50-4:50

Session 3: Division of Cancer
Advisory Board: Prof. Tadashi Yamamoto, Japan
Chair: Jun-ichi Fujisawa, Koichi Tomoda

3:50-4:05 Yosiaki Sonoda (Stem Cell Biology)
Prospectively isolated novel PDGFR and Sca-1 double positive mouse dental pulp
stem cells exist in the tooth-forming niche
4:05-4:20 Jun-ichi Fujisawa (Viral Oncology)
Analysis of ATL development in HTLV-1 infected humanized mouse model
4:20-4:35 Masanori Yamada (Surgery)
Application of MALDI-imaging mass spectrometry to esophageal and gastric cancer
research
4:35-4:50 Tomoya Katakai (Molecular Genetics, IBS)
Live imaging of high-speed lymphocyte migration in lymph node stromal network by
two-photon microscopy

4:50-5:00

Closing Remarks

Hiroo Ueno

36

The 2nd Annual Meeting of Molecular Imaging Center of Diseases
on June 15, 2013 at 4th-floor Conference Room, Hirakata Campus

Program
10:00-10:10 Opening remarks

Seiji Ito

Introduction of advisory board members

10:10-11:10 Session 1: Division of Metabolic Disorders
Advisory Board: Dr. John A. Hanover, USA
Chair: Tomoyuki Nakamura, Tomoki Ito
10:10-10:30 Masami Kageshima (Biophysics)
Atomic force microscopy – a versatile tool for imaging and probing microscopic
mechanical properties of soft materials
10:30-10:50 Satoshi Matsuda （Bioinformatics, IBS）
Immune regulation through the mTORC1 pathway
10:50-11:10 Takaya Tsuno (Radiology)
Analysis of JAK-STAT and cell death pathways induced by IFN- in humanadherent cancer cells

11:10-12:10 Session 2: Division of Neuroscience
Advisory Board: Prof. Min Zhuo, Canada
Chair: Tetsuo Sugimoto, Hirofumi Kusaka
11:10-11:25 Satoshi Kaneko (Neurology)
Activation of TGF/Smad signaling reduces aggregate formation of mislocalized
TDP-43
11:25-11:40 Masaki Isoda (Cognitive Neuroscience)
Two separate neural systems for understanding other’s actions
11:40-11:55 Kiichi Hirota (Anesthesiology)
Visualization of “hypoxia” in vivo: imaging of HIF-1 activation
11:55-12:10 Ikuko Yao (Molecular and Functional Biology)
Molecular imaging for diseases using mass spectrometry

13:00-14:00 Session 3: Division of Cancer
Advisory Board: Prof. Tadashi Yamamoto, Japan
Chair: Jun-ichi Fujisawa, Yoshiaki Sonoda
13:00-13:20 Morihiko Ishizaki (Surgery)
Clinical applications of indocyanine green-fluorescence imaging to liver surgery
13:20-13:40 Yoshihiro Ueda (Molecular Genetics, IBS)
Mst1 regulates integrin-dependent thymocyte trafficking and antigen recognition in
the thymus
13:40-14:00 Ryusuke Nakatsuka (Stem Cell Biology)
Development of a highly efficient method for isolating bone-derived small stem
cells identified in adult mouse bone

14:00-14:15 Closing Remarks

Hiroo Ueno
-37-

Evaluation by advisory board members
Evaluation sheet
Name: John Hanover
Division in charge: Metabolic Disorders
Evaluation:
1.

a. excellent, b. good, c. ordinary, d. bad,

Molecular Imaging Center of Diseases
1) Organization including management2) Objective and concept
3) Outcome
4) Prospecte. unknown

(a)
(a)
(b)
(a)

Comments
The center’s organization, goals and prospects are all extremely timely and well organized. As
the process matures and projects develop, excellent outcomes are expected for the center’sinvestigators.
2.

Division of Metabolic Disorders

Masami Kageshima (Biophysics)
Atomic force microscopy – a versatile tool for imaging and probing
1) Presentation(b)
Dr. Kageshima has explored the mechanical properties measured through viscoelasticity. This
allows measurement of the elasticity of single polymer chains and mechanical denaturation of thechains. He described methods to discriminate forces using cyclic force applications. He firstchecked a stable alpha helix. Viscoelasticity was the most informative measurement producing aviscoelasticity spectrum. His method was applied to a dextran chain and the solvation of watermolecules on a mica surface. The methods developed may have applications in Disease imaging.
Support for such emerging technologies is encouraged although the current applications andoutcomes of the work are limited. Overall, a clear presentation and substantial promise for future
outcomes.
2) Prospect(b)
Satoshi Matsuda （Bioinformatics, IBS）
Immune regulation through the mTORC1 pathway
1) Presentation(a)Dr. Matsuda has found that p85 alpha plays a pivotal role in Th17 differentiation, but to Th1 or
Th2. He found that mTORC1/Raptor mTORC2/Rictor responds to PI3K and that Rapamycin inhibits
Th17 differentiation. Th17 differentiation is impaired in Raptor deficient T cells while activationof the pathway leads to enhanced Th17 differentiation. Using Rag2-/- minus cells Th17 was
reduced by rapamycin treated mice. He argues that mTorc1 regulates Th17 differentiation via
p70S6K. Blockade of PI3K mTORC1 pathway perturbs nuclear translocation of RORgamma. He
suggests that S6K2 retains RORgamma in the nucleus. This pathway is an interesting finding
suggesting that the Th17 response may be regulated independently of Th1 and Th2 suggesting-38-

numerous therapeutic applications.
future applications.
2) Prospect(a)

In summary, a nice presentation with findings that suggest

Takaya Tsuno (Radiology)
Analysis of JAK-STAT and cell death pathways induced by IFN- in human adherent cancer cells
1) Presentation(b)
Dr. Tsuno analyzed the molecules in IFN- pathways; his goal was to in vivo verify the targetedcancer therapy developed from the in vitro studies. IFNAR1/2 and IRF9 RNAi inhibited AP
activity of IFN- in OVCAR3 cells. Trail protein was secreted onto the membrane of OVCAR3
cells in response to IFN-. Caspase 8 inhibitor restored the viability suggesting that Caspase
8/BID is important. BID may important for mitochondrial membrane potential. BID overexpression
has the potential to enhance the AP activity of IFN-. Dr. Tsuno Will use integrin with RGD in
matrices to improve the therapeutic potential. BID transfection led to mitochondrial apoptosis as
well as A549 cells. The presentation was nicely prepared and the future applications were wellfleshed out and clear.
2) Prospect(a)

Evaluation sheet
Name: Min Zhuo
Division in charge: Neuroscience
Evaluation:a. excellent, b. good, c. ordinary, d. bad,

1. Molecular Imaging Center of Diseases
1) Organization including management2) Objective and concept
3) Outcome
4) Prospecte. unknown

(a)
(a)
(a)
(a)

2. Division of Neuroscience
1) Presentation(a)2) Prospect(a)
3. General comments and suggestions
I was very impressed by individual reports presented. I think that all speakers gave excellent
talks, and the qualities of science are high. I think that one major area that can be improved infuture will be integrative research activities, or some of joint projects among individual
investigators.

Evaluation sheet
Name: Tadashi Yamamoto
Division in charge: Cancer
Evaluation:a. excellent, b. good, c. ordinary, d. bad,-39-

e. unknown1. Molecular Imaging Center of Diseases
1) Organization including management2) Objective and concept
3) Outcome
4) Prospect

(a)
(a)
(b)
(a)

Comments
The new Hirakata campus provides opportunity of interaction between basic medical scienceand clinics. This Molecular Imaging Center of Diseases should play important roles in suchinteraction. Indeed by seeing activities presented in the 2nd annual meeting, I am confident to thesuccess of the programs of the Imaging Center, as the Center integrates well both basic and clinicalResearch. The research outcome is good but probably more to come in near future.
2. Division of Metabolic Disorders
1) Presentation(a)2) Prospect(b)
3. Division of Neuroscience
1) Presentation(a)2) Prospect(b)
4. Division of Cancer
1) Presentation
2) Prospect

(a)
(b)

Besides presented, I found Dr. Ishizaki is quite active in publication. Dr. Ueda’s work on the roleof Mst1 is of high quality. He is also active in publication. Stem cell-isolation presented by Dr.
Nakatsuka would be useful.
5. General comments and suggestions
As mentioned above, the topics presented in the meeting included both clinical and basic
researches. This indicates that the Molecular Imaging Center of Diseases of KMU is well organizedaiming at mingling of researches in various departments. The quality of presentation impressed methat the center is moving actively and its future is promising. I also found that the speakers wererelatively young, which is a good sign suggesting future development. On the other hand, I wasslightly disappointed by the relatively small numbers of audience. I assume that this may be because
the meeting was held on Saturday. But please keep paying efforts in recruiting young active graduate
students and postdoctoral fellows.
I didn’t have a chance to see the facilities of imaging. High performance microscopies would bevitally important for the center. Technics of imaging mass spectrometry is useful and needs to befurther improved. Experts for the use of super-resolution microscopy and multi-photon microscopy
and EM would be necessary.
The works presented at the meeting are excellent and especially the presentation by Ueno at theclosing is quite impressive. Probably there may be other PIs whose research activities were notrepresented at the meeting. By being presented, it would be easier to see the prospect of each
Division.
-40-

Supplementary Information

資料

病態分子イメージングセンター組織運営規則
(設置)
第1条
(目的)
第 2 条

(部門)
第3条
(1)
(2)
(3)
(職員)
第4条

関西医科大学専門部に病態分子イメージングセンター(以下「センター」という。)を置く。
センターは、基礎と臨床のトランスレーショナル研究発展のため、神経、がん、代謝の 3 研究グループと支援グループの研究体制を構築する。
分子イメージングシステムを駆使して、患者と動物モデルの疾患の病態を分子から個体まで体系的に解明し、課題とする疾患の診断治療法の開発に結びつけることを目的とする。
センターに次の部門を置く。
神経部門
がん部門
代謝部門

センターの教職員は本学の教職員をもつて充て、兼務とする。ただし、本学教職員以外の者を嘱託として委嘱することができる。
(センター長)第 5 条 センターにセンター長を置く。
2 センター長は、センターを管理し、その運営にあたる。
3 センター長は、大学院医学研究科委員会構成員の互選により選出し学長が任命する。
4 センター長の任期は 2 年とし、再任を妨げない。
(副センター長)
第 6 条 センターに副センター長を置く。
2 副センター長は、センター長を補佐し、センター長不在のときは、その職務を代行する。
3 副センター長は、大学院医学研究科委員会構成員の互選により選出し、学長が任命する。
4 副センター長の任期は 2 年とし、再任を妨げない。
(部門統括者)
第 7 条 第 3 条の部門に部門統括者(基礎系・臨床系)をおく。
2 部門統括者は、センター長と副センター長とが協議して大学院医学研究科委
員会構成員の中から推薦し、学長が任命する。
(運営委員会)
第 8 条 センターに運営委員会を置く。
2 運営委員会は、次の者をもつて構成する。
(1) センター長-41-

(2) 副センター長(3) 部門統括者 6 名
3 前項第 1 号及び第 2 号に掲げる委員の任期は、在任期間とする。
4 第 2 項第 3 号に掲げる委員の任期は 2 年とする。ただし、再任を妨げない。
5 運営委員会は、センターの円滑な運営をはかるために、次の事項を審議する。
(1) センターの予算の審議(2) センター長が必要とする事項(3) センターにおける研究の充実並びに促進に関する事項(4) 研究の成果の活用に関する事項(5) その他センター運営に関する事項
6 センターの施設利用に関することは、綜合研究施設に付託する。
7 運営委員会は、必要に応じてセンター長がこれを招集し、議長となる。
8 運営委員会の庶務は、学部事務部研究課において行う。
(規則の改廃)第 9 条 この規則の改廃は、センター運営委員会の審議を経て、大学院医学研究科委員会の承認を得なければならない。
附 則
この規則は、平成 23 年 8 月 1 日から施行する。
センター設置初年度の運営委員会構成員任期の終期は、平成 25 年 3 月 31 日と
する。

-42-

The 1st Annual Meeting of Molecular Imaging Center of Diseases
on May 1, 2012 at 13th-floor Auditorium, Hirakata Hospital

Program
1:00-1:10
1:10-1:30

1:30-2:30

Opening remarks
Introduction of advisory board members
Outline and prospect of Molecular Imaging Center of Diseases
Seiji Ito
Session 1: Division of Metabolic Disorders
Advisory Board: Dr. John A. Hanover, USA
Chair: Tomoyuki Nakamura, Kazuichi Okazaki

1:30-1:45 Tomoyuki Nakamura (Molecular Pharmacology)
Development and regeneration of elastic fiber
1:45-2:00 Hiroyasu Tsukaguchi （Internal Medicine 2）
Genetic approaches to identify new targets of molecular imaging for kidney andneurodevelopmental disorders
2:00-2:15 Toshiro Fukui (Internal Medicine 3)
The specific linker phosphorylation of Smad2/3 indicates epithelial stem cells incolon; particularly increasing in the regeneration phase of murine model of dextran
sulfate sodium-induced colitis
2:15-2:30 Tomoki Ito (Internal Medicine 1)
Dendritic cells as therapeutic targets of autoimmune diseases.

2:30-3:50

Session 2: Division of Neuroscience
Advisory Board: Prof. Min Zhuo, Canada
Chair: Tetsuo Sugimoto, Hirofumi Kusaka

2:30-2:42 Masayoshi Okada (Cellular and Molecular Physiology)
The degradation of the inwardly rectifying potassium channel, Kir2.1, depends on thecurrent level
2:42-2:54 Taketoshi Wakabayashi (Anatomy and Cell Science)
Cell type specific expression of lamin subtypes in rat retinal neurons
2:54-3:06 Hakuo Takahashi (Clinical Sciences and Laboratory Medicine)
Analysis of the brain digitalis-like substance underlying hypertension
3:25-3:37 Satoshi Kaneko (Neurology)
Analysis of intracytoplasmic inclusions of neurodegenerative diseases by imaging
mass spectrometry
3:37-3:50 Masahito Shimojo (Molecular and Functional Biology)
The challenges of neurodegenerative disease modeling using patient-specific iPSCs-43-

3:50-4:50

Session 3: Division of Cancer
Advisory Board: Prof. Tadashi Yamamoto, Japan
Chair: Jun-ichi Fujisawa, Koichi Tomoda

3:50-4:05 Yosiaki Sonoda (Stem Cell Biology)
Prospectively isolated novel PDGFR and Sca-1 double positive mouse dental pulp
stem cells exist in the tooth-forming niche
4:05-4:20 Jun-ichi Fujisawa (Viral Oncology)
Analysis of ATL development in HTLV-1 infected humanized mouse model
4:20-4:35 Masanori Yamada (Surgery)
Application of MALDI-imaging mass spectrometry to esophageal and gastric cancer
research
4:35-4:50 Tomoya Katakai (Molecular Genetics, IBS)
Live imaging of high-speed lymphocyte migration in lymph node stromal network by
two-photon microscopy

4:50-5:00

Closing Remarks

Hiroo Ueno

-44-

Abstract
Outline and prospect of Molecular Imaging Center of Diseases
Seiji Ito (Molecular and Functional Biology)
Molecular Imaging Center of Diseases was established in September 2011 upon adoption of the
program for the strategic research foundation at private universities supported by the Ministry of
Education, Culture, Sports, Science, and Technology (MEXT) of Japan. The Center aims tocontinue the former 5-year project “Interactive translational research toward the treatment of
intractable neural diseases by restoration and regeneration” carried out in Brain Medical ResearchCenter at Takii campus in 2006－2011 and expand translational research between basic and clinicaldepartments in a wide-range field of medicine in Hirakata new campus which will open in April
2013. We plan to establish the Center on the 5th floor of the mid-rise building in Hirakata new
campus and endeavor to make it a research foundation capable of pursuing interdisciplinary andstrategic research. The objectives of the Center are (1) to systematically elucidate pathogenesis ofvarious diseases by use of animal disease models and patient samples from molecular to individuallevels, (2) to translate the basic research on diseases into clinical application, by making the most of
state-of-the-art imaging systems including two-photon microscopy and imaging mass spectrometry,and (3) to cultivate young researchers. To accomplish them, the Center consists of three divisionsof Neuroscience, Cancer, and Metabolic Disorders, each of which is managed by two division heads
assigned from basic and clinical fields and a supporting division. The supporting division helps
project members to promote research activities and cultivate young researchers. The research
activities will be evaluated by advisory board members from outside. I will introduce examples ofcooperative research and overview the prospect of the Center.

Session 1

Division of Metabolic Disorders
Development and regeneration of elastic fiber
Tomoyuki Nakamura (Molecular Pharmacology)

Elastic fibers are ubiquitous extracellular matrix (ECM) components responsible for tissue
elasticity. Degradation of elastic fibers causes emphysema, arterial stiffness and loose skin. The
turnover rate of elastic fibers is very low, and there is no method to effectively regenerate elastic
fibers. The process of elastic fiber assembly involves elastin monomer (tropoelastin) deposition on
microfibrils, followed by cross-linking of tropoelastin by lysyl oxidase. As the turnover rate ofelastic fibers are very low, elastic fiber regeneration has been considered to be difficult.
We found that at least three secreted proteins, fibulin-4 and -5, and LTBP-4 (latent
TGFβ-binding protein 4), play essential roles in the elastogenesis process. Mouse arteries that lackeither of these genes were stiff, and the elastic lamellae were disorganized. We show independentroles of fibulin-4 and -5 in elastic fiber assembly that do not overlap each other, and an
LTBP-4-dependent role of fibulin-5.

Genetic approaches to identify new targets of molecular imaging forkidney and neurodevelopmental disorders.Hiroyasu Tsukaguchi (Internal Medicine 2)
Chronic kidney diseases and neurodevelopmental diseases (i.e., mental retardation, epilepsy)
are major health concerns with an overall prevalence of ~ 1-2% worldwide. The development ofsimpler, non-invasive molecular imaging techniques, visualizing the spatio-temporal progression of
disease processes, will greatly stimulate the generation of new therapeutic strategies for these-45-

common disorders. In this study, we reasoned that understanding the pathophysiological basis of raremendelian disorders is the best way to select novel target molecules suitable for imaging studies inmore common disorders.
To address this issue, as a first step, we searched as-yet-undefined genes causing a rarecongenital disorder of Galloway-Mowat syndrome (GMS, MIM 251300). GMS is characterized byco-occurrence of malformation in central nerve systems (CNS) and renal glomeruli. Clinically, GMS
patients typically exhibit two core manifestations consisting of (1) nephrotic syndrome progressing
to end stage renal disease, and (2) psychomotor retardation and/or epilepsy. In ten Asian GMS
patients, we screened the exomes and structural variations across the whole genome. Several
suspicious candidate genes are now on the way of being validated for their biological significance.
To gain more insights into the pathophysiology underlying GMS, we generated patient-specific iPS
cells from one family (3 clones under 12 passages, in collaboration with Prof. Era’s lab, Kumamoto
University).
A neuron and glomerular podocyte share a common biological feature of terminallydifferentiated cells that exert their function through formation of specialized cell-cell junctions (i.e.,
synapse and slit diaphragm). Therefore, diseases pathways underlying GMS, e.g., metabolic activity,
cell proliferation, apoptosis, and migration, etc. are likely shared by these two cellular lineages.
Identification of GMS genes will open new avenue to create a real-time, noninvasive imaging
system that detect an unique biological process indispensable for building up a proper tissue
architecture of renal glomeruli and CNS.

The specific linker phosphorylation of Smad2/3 indicates epithelial stem
cells in colon; particularly increasing in the regeneration phase of murine
model of dextran sulfate sodium-induced colitis
Toshiro Fukui (Internal Medicine 3)
Stem cells play roles in mucosal cell homeostasis and repair. Although they are supposed to belocated around the crypt base of colon, useful markers have not been elucidated. Previously, we haveidentified the significant expression of Smad2/3, phosphorylated at specific linker threonine residues(pSmad2/3L-Thr), in specific epithelial cells of murine stomach and have suggested these cells to bestem cells. (J Gastroenterol 2011:46:456-68) In the present study, we explored whether
pSmad2/3L-Thr could serve as a marker for stem cells of colon.
Normal colons from C57BL/6 or Lgr5-EGFP knock-in (Lgr5-EGFP-IRES-CreERT2) mice andcolons from murine model of dextran sulfate sodium (DSS)-induced colitis were examined. In
normal colons, pSmad2/3L-Thr immunostaining-positive cells were found around the crypt base anddetected between Ki67 immunostaining-positive cells, but immunohistochemical co-localization of
pSmad2/3L-Thr with Ki67 was never observed. pSmad2/3L-Thr immunostaining-positive cells
showed co-localization with cytokeratin8 and CDK4, different localization with ChromograninA,and similar localization with doublecortin and calcium/calmodulin-dependent protein kinase-like-1(DCAMKL1) immunostaining-positive cells. In DSS-induced colitis, both pSmad2/3L-Thr and Ki67
immunostaining-positive cells were significantly increased in the regeneration phase and decreasedin the injury phase compared with those of normal C57BL/6 mice. In colon of Lgr5-EGFP knock-in
mice, most pSmad2/3L-Thr immunostaining-positive cells showed co-localization with Lgr5 (GFP),
but a small number of pSmad2/3L-Thr immunostaining-positive cells showed different localization
with Lgr5. After immunofluorescent staining, we stained the same sections with hematoxylin-eosin
and observed pSmad2/3L-Thr immunostaining-positive cells indicated undifferentiatedmorphological features and were confirmed around the crypt base. 5-bromo-2-deoxyuridine (BrdU)
labeling assay was used to examine label-retention (multiple BrdU pulses; three times a day for 2
days)ofpSmad2/3L-Thr
immunostaining-positive
cells.
Some
pSmad2/3L-Thr
immunostaining-positive cells showed co-localization with BrdU at 5 d, 10 d, and 15 d after BrdU
-46-

administration.
We have identified the significant expression of pSmad2/3L-Thr in specific epithelial cells ofmurine colon and have suggested these cells to be stem cells.

Dendritic cells as therapeutic targets of autoimmune diseases
Tomoki Ito (Internal Medicine 1)
Dendritic cells (DCs) are widely distributed in the body to enhance the innate immune responseby cytokine production through antigen recognition and initiate acquired immunity by priming naïve
CD4+ T cells. Therefore, DCs are the master cells in activating immune responses. Plasmacytoid
dendritic cells (pDCs), one of the human DC subsets, play not only a central role in antiviral immune
response in innate host defense but also a pathogenic role in the development of the autoimmune
responses by vicious spiral based on the dysregulated type I IFN production through sensing immune
complexes containing self-nucleic acids. Thus, type I IFNs and pDCs represent key molecular andcellular pathogenic components in autoimmune diseases such as systemic lupus erythematosus(SLE). Accordingly, control of the dysregulated pDC-derived type I IFN production provides analternative treatment strategy for SLE. We focused on two agents, IB kinase inhibitor(BAY11-7082) and HMG-CoA reductase inhibitors (statins), and investigated theirimmunomodulatory effects in targeting the IFN response on pDCs.
Our study identified that both BAY11 and statins have the ability to inhibit nuclear translocationof IRF7 and IFN- production in human pDCs. We also found that these agents inhibited both invitro IFN- production induced by the SLE serum and the in vivo serum IFN- level induced byinjecting mice with TLR ligand. These findings suggest that BAY11 and statins have the therapeuticpotential to attenuate the IFN environment by regulating the pDC function and thus provide thenovel foundation for the development of an effective immunotherapeutic strategy against
autoimmune disorders such as SLE.

Session 2

Division of Neuroscience

The degradation of the inwardly rectifying potassium channel, Kir2.1,
depends on the current level
Masayoshi Okada (Cellular and Molecular Physiology)
Protein expression is regulated by synthesis as well as degradation. Inwardly rectifying K+
channel (Kir2.1) regulates the excitability of nervous, cardiac, and vascular smooth muscular cells,
and its expression level is accurately regulated. In fact, both loss- and gain-of-function mutations of
Kir2.1 result in arrhythmia. But little is known about the degradation of Kir2.1. To examine thedegradation of Kir2.1, we constructed Kir2.1 fusion proteins with SNAP-tag and fluorescent timer(FT). The SNAP-tag, which covalently binds to a specific membrane permeable fluorescent dye,allows us to conduct a pulse-chase experiment with fluorescence. Our pulse-chase experiments
revealed the current-dependent degradation of Kir2.1 by following results: 1) shorter half-life of
SNAP-Kir2.1 in high expression cells; 2) elongation of half-life by the blockade of Kir current; and3) shorter half-life in a hyper-conductive mutant of Kir2.1. The fluorescence of FT changes
spontaneously and slowly from green to red; thereby the green/red ratio allows us detect the changes
in the half-life of the FT-fusion protein. Expectedly, the green/red ratio of FT-Kir2.1 was decreasedby the channel blockade, confirming the current-dependent degradation. Our results suggest apresence of K+ sensor and the usefulness of these two methods, which are in vivo applicable and do
not need radioisotope or antibody, in studying neurodegenerative diseases.
We also introduce the outlines of other ongoing projects.
-47-

1.
2.
3.
4.
5.
6.
7.
8.

Compensation for the reduced activity in the hippocampal newborn neurons.
Enhancement of the titer of lentiviral vectors, which express K+ channels.
High-though-put assay for the Kir2.1 using GFP.
Screening for Kir blocker and opener in Mexican Scorpion venom.
Lenti- and Retro-viral vectors specific for somatotroph.
Ion channels involved in the secretion of HCO3- from pancreatic duct cell.
Lentiviral vector specific for the secretin receptor expressing cells.
Ca2+ imaging and plasticity of the hippocampal GABAergic synapses.

Cell type specific expression of lamin subtypes in rat retinal neurons
Taketoshi Wakabayashi (Anatomy and Cell Science)
The nuclear lamins are type V intermediate filament proteins and the major component of thenuclear lamina. They are grouped into two subtypes, A-type, which include lamins A and C, andB-type, which include lamins B1 and B2. Lamins support nuclear membrane mechanically and arealso essential for proper cellular functions, including DNA replication, gene expression anddifferentiation. Abnormal expression of lamins causes several inherited diseases including muscular
dystrophy and neuropathy. However, the expression patterns of lamin subtypes have not been wellstudied, especially in the nervous system. In our laboratory, we have reported the expression patternsof lamin subtypes in neurons during adult neurogenesis.
Retina belongs to the central nervous systems. Because of its well-organized cellulararchitecture and relative simplicity, the retina has been used as a representative model for studying
neuronal cells in the central nervous system. Here, we report the immunohistochemical expression
patterns of lamin subtypes in retinal neurons of adult rat. All retinal neurons expressed lamin B1 andB2 in relatively equal amounts. By contrast, the expressions of A-type lamins were differentiallyregulated depending on the cell types. Photoreceptor cells did not show any immunoreactivity for
anti-lamins A and C antibodies. Other retinal neurons, which include retinal ganglion cells, amacrine
cells, bipolar cells and horizontal cells, were all positive for lamin C. Lamin A is expressed only inhorizontal cells and small populations of retinal ganglion cells. The differential expression pattern oflamin subtypes in retinal neurons suggests that they may be involved in cellular differentiation andexpression of cell-specific genes in individual retinal neurons.

Analysis of the brain digitalis-like substance underlying hypertension
Hakuo Takahashi (Clinical Sciences and Laboratory Medicine)
The central nervous system has a key role in regulating the circulatory system by modulating the
sympathetic and parasympathetic nervous systems, pituitary hormone release, and the baroreceptor
reflex. Digoxin- and ouabain-like immunoreactive materials were found more than 20 years ago in
the hypothalamic nuclei. These factors appeared to localize to the paraventricular and supraoptic
nuclei and the nerve fibers at the circumventricular organs and supposed to affect electrolyte balanceand blood pressure. The turnover rate of these materials increases with increasing sodium intake. As
intracerebroventricular injection of ouabain increases blood pressure via sympathetic activation, an
endogenous digitalislike factor (EDLF) was thought to regulate cardiovascular system-related
functions in the brain, particularly after sodium loading. Experiments conducted mainly in rats
revealed that the mechanism of action of ouabain in the brain involves sodium ions, epithelial
sodium channels (ENaCs), the renin–angiotensin–aldosterone system (RAAS) and oxidative stress,
all of which are affected by sodium loading. Rats fed a high-sodium diet develop elevated sodium
levels in their cerebrospinal fluid, which activates ENaCs. Activated ENaCs and/or increased
intracellular sodium in neurons activate the RAAS; this releases EDLF in the brain, activating the
sympathetic nervous system. The RAAS promotes oxidative stress in the brain, further activating the-48-

RAAS and augmenting sympathetic outflow. Angiotensin II and aldosterone of peripheral origin act
in the brain to activate this cascade, increasing sympathetic outflow and leading to hypertension.
Thus, the brain Na+–ENaC–RAAS–EDLF axis activates sympathetic outflow and has a crucial rolein essential and secondary hypertension. Major antihypertensive agents available recently are allacting at the central sites to reduce sympathetic activity and blood pressure. However, the exact siteof actions and the cascade operating in the brain are left undetermined. Our project aims toanatomically visualize the digitalislike substance and the related bioactive materials in the brain to
elucidate the mechanism of actions in hypertension.

Analysis of intracytoplasmic inclusions of neurodegenerative diseases
by imaging mass spectrometry
Satoshi Kaneko (Neurology)
The presence of insoluble protein aggregates in the cytoplasm of neurons is a key pathologicalfeature of most neurodegenerative diseases. In several disorders, the disease-specific pathologicalproteins were identified. However, the normal and pathological functions of these proteins arelargely unknown. Through this project we are planning to analyze biomolecules in relation to
intracytoplasmic inclusions using imaging mass spectrometry (IMS). Prior to analysis of humanbrain section by IMS, we performed immunohistochemical studies on intacytoplasmic inclusions ofneurodegenerative diseases to obtain further preliminary findings.
1) We investigated a family manifesting amyotrophic lateral sclerosis (ALS) with aheterozygous E478G mutation in the optineurin (OPTN) gene. TAR DNA-binding protein 43(TDP-43)-positive neuronal intracytoplasmic inclusions were reactive with anti-ubiquitin andanti-p62 antibodies, but negative for anti-OPTN antibody. Immunoreactivity of nuclear TDP-43 was
reduced in inclusion-bearing cells. Fragmentation of Golgi apparatus was identified in 70% of theanterior horn cells. The presence of anterior horn cells with preserved nuclear TDP-43 and a
fragmented Golgi apparatus indicates that patients with the E478G OPTN mutation would manifest
fragmentation of Golgi apparatus before loss of nuclear TDP-43.
2) Immunohistochemical analysis revealed that all the basophilic inclusions (BIs) were positive
for OPTN, Fused in sarcoma (FUS) and myosin VI in sporadic basophilic inclusion body disease(BIBD) patients and familial ALS with FUS mutation. However, the BIs showed no
immunoreactivity for TDP-43 or SOD1. Therefore, OPTN associates with each of the 3 major
ALS-related proteins, i.e., TDP-43, SOD1, and FUS. FUS is known to act as a co-activator of
NF-B, and OPTN negatively regulates NF-B activation. Therefore, sequestration of both FUS andOPTN in BIs may induce dysregulation of NF-B activation, leading to neurodegeneration.
Moreover, OPTN and myosin VI play a role in the maintenance of Golgi apparatus. Theirsequestration in BIs may be an underlying pathomechanism of fragmented Golgi apparatus.
3) We demonstrated for first time that Smad ubiquitination regulatory factor-2 (Smurf-2) isco-localized in the phosphor-tau inclusions in progressive supranuclear palsy (PSP). As Smurf-2 isan E3 ligase of Smad2, intracellular signaling component of TGF, dysregulation of TGF/Smadsignaling may be an underlying pathomechanism of PSP.
These findings suggest that fragmentation of Golgi apparatus, impairment of the RNA quality
control system due to mislocalized RNA binding proteins, dysregulation of NFB activation and/or
disturbed TGF/Smad signaling may take part in pathological processes of neurodegeneration.

The challenges of neurodegenerative disease modeling using
patient-specific iPSCs
Masahito Shimojo (Molecular and Functional Biology)-49-

Neurodegenerative diseases such as Huntington’s disease (HD), Alzheimer’s disease, andamyotrophic lateral sclerosis are progressive diseases involving the degeneration of selected neurons.
Molecular biological analyses are advancing our understanding of intractable neurodegenerative
diseases, but translating them to the treatment of diseases is slow. For example, HD is a choreic
psychiatric neurodegenerative disease caused by a mutation in the huntingtin protein (Htt) in which
expansion of CAG tract at the 5’-end of the gene is translated into a toxic polyglutamine (polyQ)
stretch. The pathogenic mechanisms induced by polyQ-Htt are still not clearly understood and lackadequate models to investigate. It has been reported that Htt interacts with a key transcription factor,
RE1-silencing transcription factor (REST). REST is a major regulator of many neuronal genes
repressing their expression. The interaction between Htt and REST was found to retain REST in thecytosol, thereby preventing REST target gene repression. Several lines of evidence indicate thatpolyQ-Htt increases transcriptional repression by REST in HD. This results in increased occupancy
of multiple RE1s in different genes and would cause a number of REST target microRNAs andneuronal genes to be aberrantly repressed, which now provides us with a target for developing
treatments for HD. However we are lacking adequate systems for further advancing these studies inhuman-derived material.
The development of therapeutic strategies for treating diseases has relied heavily on the use ofanimal models. However they are limited in their ability to recapitulate the human disease. Recent
molecular technologies could provide human materials for studying human disease throughdifferentiating human stem cells into disease-specific cell types; especially disease-specific induced
pluripotent stem cells (iPSCs). The iPSCs can be generated by reprogramming fibroblasts derived
from individuals with a specific neurodegenerative disease. Generation and characterization of
iPSCs from patients have real advantages due to their ability to be differentiated into specific cell
types such as neurons and retain the defect causing the disease. In neurodegenerative diseases, such
affected cells cannot be collected from patients but can be generated from disease-specific iPSCs. By
using iPSCs for disease modeling, the genetic information contributing to neurodegeneration, isretained in in vitro studies.
Thus the generation of disease-specific iPSCs from patients with currently incurable diseases is
a promising approach for studying the mechanism of a particular disease at the cellular andmolecular levels and for developing new drugs and therapeutic targets.

Session 3

Division of Cancer

Prospectively isolated novel PDGFR and Sca-1 double positive mousedental pulp stem cells exist in the tooth-forming niche
Yoshiaki Sonoda, (Stem Cell Biology and Regenerative Medicine)
Mouse incisors erupt continuously throughout the life of the animal. Tooth-derived stem cells
are thought to exist in the apical end of the mouse incisor to mediate this process. Dental pulp stem
cells (DPSCs) are considered to be one of the tooth-derived stem cells. The isolation of DPSCs wasformerly carried out by cell attachment to cell culture dishes, thus resulting in the isolation of
unpurified heterogeneous DPSCs. On the other hand, a previous study showed that mouse bonemarrow-derived mesenchymal stem cells (MSCs) could be prospectively isolated by fluorescence
activated cell sorting (FACS) using MSC markers. In this study, we successfully isolated mouse
DPSCs from mandibular incisors by FACS using the MSC markers, PDGFRa and Sca-1. MousePDGFRa and Sca-1 double positive DPSCs exhibited a high growth potential in vitro and in vivo. In
addition, the DPSCs expressed odontoblast progenitor cell markers and they showed the ability todifferentiate into osteocytes, chondrocytes and adipocytes. An immunofluorescence analysis of the-50-

apical end of mouse incisors revealed that PDGFR+Sca-1+ cells are present in the cervical loop(CL) of the incisors and the region surrounding the CL. In conclusion, these results revealed that themouse PDGFR+Sca-1+ DPSCs possess the potential to differentiate into osteocytes, chondrocytes
and adipocytes. Furthermore, our findings suggest that PDGFR+Sca-1+ DPSCs exist in the niche
for tooth-forming cells, because these cells localized in the CL of the incisors, and exhibited highgrowth potential.

Analysis of ATL development in HTLV-1 infected humanized mouse model
Jun-ichi Fujisawa (Viral Oncology)
Adult T-cell leukemia (ATL) is an aggressive malignancy of mature CD4+ CD25+ T cells that
affects about 5% of individuals infected with HTLV-1 thirty to fifty years after neonate infection.
The molecular and genetic factors induced by HTLV-1 during such a long latent period remain
unclear, in part from the lack of an animal model that accurately recapitulates leukemogenesis. To
understand the mechanism of ATL development, we established a mouse model of HTLV-1
infection by using the humanized mouse established by the intra-bone marrow transplantation of
NOG-SCID mouse (IBMI-huNOG) with CD133+ hematopoietic stem cells purified from human
cord blood.
Human T-lymphocytes differentiate normally in the spleen and the peripheral blood of
IBMI-huNOG mice in several months after transplantation and peritoneal injection of HTLV-1
producing cells resulted in the leukemic growth of CD4+ CD25+ T-lymphocytes and led to thehepatosplenomegaly three to four months after infection. In the late period of infection, cells withhighly lobulated or flower-shaped nuclei appeared in the peripheral blood as seen in ATL patient.
Oligoclonal expansion of infected T cells was observed and common T-cell clones withidentical integration sites were identified in CD25- and CD25+ populations, indicating theconversion from CD25- to CD25+ in infected CD4+ T-cell populations in the course of selective
growth. Although Tax protein of HTLV-1 has been thought to be associated with theleukemogenesis, expression of Tax was observed in CD25- population but not in CD25+
population. It was therefore suggested that Tax might enhance the proliferation of CD25- CD4+
T-lymphocytes in early phase of infection but that some epigenetic change should be involved in
the outgrowth of CD25+ CD4+ T-lymphocytes in the late phase.
Thus, the HTLV-1 infected huNOG mouse model should provide a valuable system for the
analysis of ATL pathogenesis and the development of treatments against various HTLV-1
associated diseases.

Application of MALDI-imaging mass spectrometry to esophageal andgastric cancer research
Masanori Yamada (Surgery)
Acute abdomen obstruction is a common disease but may threat life occasionally. In thepresent case, she had severe abdominal pain and deteriorated rapidly in a shorter period.
Abdominal CT scanning demonstrated a high density mass in the dilated small intestine and air-fluid
level formation without free air. We did not know what caused acute abdomen obstruction.
Laparoscopic operation was performed to excise the inflamed small intestine and the obstructive
material. For imaging MS analysis, the surgically isolated intestinal portion and the foreign body
were fixed and paraffin embedded as performed routinely at Kansai Medical University Hospital.
With direct MALDI-TOF MS imaging, we identified mochi as the obstructing material in a small
intestinal obstruction. This is the first case of identification of obstructing material in direct MS
analyses of histopathological samples. MALDI-IMS is a very rapid and accurate technique withhigh sensitivity for direct analysis of materials in histopathological samples.
-51-

The liver is a frequent site of metastasis from gastric cancer and its rate is from 5 to 10%. The
hepatic resection or re-resection is thought to be a benefit for patients if gastric cancer and its
metastasis are well controlled, and liver metastasis is solitary or restricted to one lobe. However,hepatic resection is carried out in less than 10% of patients with metastasis because of multiple
hepatic nodules or associated peritoneal dissemination. Our prospect of research in Molecular
Imaging Center of Diseases is to detect a biomarker for liver metastasis with direct MALDI-TOF
MS imaging, which can help us perform appropriate therapy for patients by early diagnosis ofmetastasis.

Live imaging of high-speed lymphocyte migration in lymph node stromal
network by two-photon microscopy
Tomoya Katakai (Molecular Genetics, IBS)
Lymph node (LN) is a small organ for immuno-surveillance filtrating and monitoring foreign
substances (antigens) in lymph drainage from peripheral tissues. Live imaging using two-photon
laser scanning microscope (TP-LSM) is a valuable technique for addressing the behavior offluorescent-labeled motile cells inside the tissues. Applying TP-LSM to live LN has revealed thatlymphocytes within the tissue parenchyma robustly migrate searching for antigens. Especially, T
cells are moving at more than 10mm/min on average velocity. Tissue structure of LN is supportedby the three-dimensional (3D) network of non-hematopoietic stromal cells, which has beensuggested to be involved in the interstitial T cell migration. However, details of the interaction
between T cells and stromal cells, as well as underling molecular mechanism for the efficient,high-speed movement of T cells remain unclear. To address these issues, we set out to establish a
TP-LSM-based imaging system using LN tissue slice. T cells showed a complicated migratory
behavior in close association with stromal network. Antibody blockage of the major lymphocyte
integrin LFA-1 and the ligand ICAM-1 partially but significantly reduced T cell motility within LN
slice. T cell migration was also decreased in LN from ICAM-1 deficient mice compared with thewild type LN. Moreover, the inhibition of LFA-1 led to T cells’ slip on and detachment fromstromal network but still left substantial motility. Taken together, our observations indicate that
LFA-1/ICAM-1-dependent and -independent motilities enables T cells to exhibit high-speed
migration within 3D tissue environment. In addition, stromal network functions as scaffold for theefficient forward movement of T cells.

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The 2nd Annual Meeting of Molecular Imaging Center of Diseases
on June 15, 2013 at 4th-floor Conference Room, Hirakata Campus

Program
10:00-10:10 Opening remarks

Seiji Ito

Introduction of advisory board members

10:10-11:10 Session 1: Division of Metabolic Disorders
Advisory Board: Dr. John A. Hanover, USA
Chair: Tomoyuki Nakamura, Tomoki Ito
10:10-10:30 Masami Kageshima (Biophysics)
Atomic force microscopy – a versatile tool for imaging and probing microscopic
mechanical properties of soft materials
10:30-10:50 Satoshi Matsuda （Bioinformatics, IBS）
Immune regulation through the mTORC1 pathway
10:50-11:10 Takaya Tsuno (Radiology)
Analysis of JAK-STAT and cell death pathways induced by IFN- in humanadherent cancer cells

11:10-12:10 Session 2: Division of Neuroscience
Advisory Board: Prof. Min Zhuo, Canada
Chair: Tetsuo Sugimoto, Hirofumi Kusaka
11:10-11:25 Satoshi Kaneko (Neurology)
Activation of TGF/Smad signaling reduces aggregate formation of mislocalized
TDP-43
11:25-11:40 Masaki Isoda (Cognitive Neuroscience)
Two separate neural systems for understanding other’s actions
11:40-11:55 Kiichi Hirota (Anesthesiology)
Visualization of “hypoxia” in vivo: imaging of HIF-1 activation
11:55-12:10 Ikuko Yao (Molecular and Functional Biology)
Molecular imaging for diseases using mass spectrometry

13:00-14:00 Session 3: Division of Cancer
Advisory Board: Prof. Tadashi Yamamoto, Japan
Chair: Jun-ichi Fujisawa, Yoshiaki Sonoda
13:00-13:20 Morihiko Ishizaki (Surgery)
Clinical applications of indocyanine green-fluorescence imaging to liver surgery
13:20-13:40 Yoshihiro Ueda (Molecular Genetics, IBS)
Mst1 regulates integrin-dependent thymocyte trafficking and antigen recognition in
the thymus
13:40-14:00 Ryusuke Nakatsuka (Stem Cell Biology)
Development of a highly efficient method for isolating bone-derived small stem
cells identified in adult mouse bone

14:00-14:15 Closing Remarks

Hiroo Ueno
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Abstract
Session 1

Division of Metabolic Disorders

Atomic force microscopy – a versatile tool for imaging and probing
microscopic mechanical properties of soft materials
Masami Kageshima (Biophysics)
Atomic force microscopy (AFM) has gained recognition as an imaging tool for surfaces of various
materials ranging from solids to soft biological materials. Its force sensor is well-suited for
spatially-resolved measurement of mechanical response in the form of a complex quantity, i.e. a
viscoelasticity. During the course of research the author has developed a wide-band magnetic
excitation AFM apparatus, with which viscoelastic response of the sample under study is obtained ina wide frequency range by exciting the force sensor with an well-controlled magnetic force. Featuresof the instrument will be briefly overviewed. By utilizing the potential of AFM it’s possible todirectly access folding/unfolding dynamics of single biopolymers through viscoelasticity. A single
polymer chain is tethered between the AFM probe apex and the substrate. As it is stretched with atensile force it undergoes structural deformation and corresponding features in its viscoelasticity
profile are obtained. Examples of peptide in α-helix form, dextran, and titin, a giant protein moleculein muscle, will be shown1). In addition to single molecules, interest is also oriented to structure anddynamics of solid-liquid interfaces, especially those relevant to life phenomena. Response of watermolecules on a hydrophilic surface will be shown. A novel pulse-response measurement techniquewill also be briefly introduced2). It should be noted that the potential of AFM as a tool for analyzingmechanical response also covers mesoscopic or macroscopic systems like cells or organisms. Proper
understanding of its imaging mechanism and, if necessary, instrumentation technique will further
enhance its applicability.
1. M. Kageshima, Curr. Pharm. Biotech. 13, 2575-2588, 2012.
2. M. Kageshima, Beilstein J. Nanotech. 3, 260-266, 2012.

Immune regulation through the mTORC1 pathway
Satoshi Matsuda (Bioinformatics, IBS)
The mammalian target of rapamycin (mTOR) is a Ser/Thr kinase that affects broad aspects ofcellular functions including immunity. mTOR is the core component of the mTOR complex 1(mTORC1), which contains the adapter protein Raptor, whereas Rictor and Sin1 classify mTOR
complex 2 (mTORC2). mTORC1 acts downstream of the PI3K-Akt pathway to phosphorylate p70
S6 kinase (S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) in a
rapamycin-sensitive manner. Although the PI3K-Akt-mTORC1 axis is activated during T cell
activation, its physiological role remains obscure. Here, we demonstrate that the suppression of thisaxis by deletion of p85a, a predominantly expressed regulatory subunit of class IA PI3K, or Raptor,
an essential component of mTORC1, impairs Th17 differentiation. We further demonstrate that S6K
is involved in the PI3K-Akt-mTORC1 axis-mediated nuclear translocation of RORg, a mastertranscriptional regulator for Th17 differentiation. These results point toward a pivotal role of thePI3K-Akt-mTORC1-S6K axis in Th17 differentiation.
1. Ohtani, M., Hoshii, T., Fujii, H., Koyasu, S., Hirao, A. and Matsuda, S. mTORC1 in intestinal CD11c+CD11b+
dendritic cells regulates intestinal homeostasis by promoting IL-10 production. J.Immunol. 188, 4736-4740, 2012.
2. Kurebayashi, Y., Nagai, S., Ikejiri, A., Ohtani, M., Ichiyama, K., Baba, Y., Yamada, T., Egami, S., Hoshii, T.,
Hirao, A., Matsuda, S., and Koyasu, S. PI3K-Akt-mTORC1-S6K1/2 axis controls Th17 differentiation by-54-

regulating Gfi-1 expression and nuclear translocation of RORg. Cell Rep. 1, 360-373, 2012).

Analysis of JAK-STAT and cell death pathways induced by IFN- in humanadherent cancer cells
Takaya Tsuno (Radiology)
Human interferon (IFN)-α has been approved for the treatment of certain types of cancer.
Nevertheless, its effectiveness has met with varying degrees of success. Currently, a number oftumors including solid carcinomas are still resistant to IFN-α. Therefore, we have investigated key
molecules playing a significant role in cell viability regulated by IFN-α.
First, in the Janus kinases/Signal Transducers and Activators of Transcription (JAK-STAT)
pathway initiating IFN-α signal, IFN regulatory factor (IRF) 9-RNA interference (RNAi) completely
restored cell viability regulated by IFN-α in human ovarian adenocarcinoma OVCAR3 cells
sensitive to IFN-α (1). IRF9-RNAi followed by IFN-α treated in OVCAR3 cells inhibited gene
expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) which is one of cell
death ligands (1). IFN-α also upregulated TRAIL protein level in OVCAR3 cell–microenvironment(2). In cell death pathway after binding TRAIL to the receptors, BH3 interacting domain deathagonist (BID)-RNAi significantly restored cell viability regulated by IFN-α in OVCAR3 cells (2).
BID-RNAi prevented IFN-α from collapsing mitochondrial membrane potential (2). Furthermore,
pIRES-hBID plasmid led to significant inhibition of cell viability regulated by IFN-α in human lungcarcinoma A549 cells resistant to IFN-α (2). Thus, our in vitro studies suggest that BID is crucial forcell viability regulated by IFN-α which can induce mitochondria-mediated apoptosis.
To assess BID overexpression for the treatment of IFN-α-resistant tumors in a living body, invivo study has also been performed. Intravenous and subcutaneous injections of pIRES-BID andpegylated human IFN-α were administered in nude mice bearing subcutaneous A549 xenografts,
respectively. Electron microscopy findings revealed mitochondrial apoptosis in A549 xenografts
treated with pIRES-BID and IFN-α. Live imaging study detecting near-infrared fluorescent tracers
also indicated apoptosis induced by pIRES-BID and IFN-α in A549 xenografts. Consequently, ourcurrent study suggests that BID is a notable potential to be a targeted therapy for IFN-α resistanttumors.
1.
2.

IRF9 is a key factor for eliciting the antiproliferative activity of IFN-α. Tsuno T, Mejido J, Zhao T, Schmeisser H,
Morrow A, Zoon KC. J Immunother. 32, 803-816, 2009.
BID is a critical factor controlling cell viability regulated by IFN-α. Tsuno T, Mejido J, Zhao T, Phillips T, Myers
TG, Bekisz J, Zoon KC. J Immunother. 35, 23-31, 2012.

Session 2

Division of Neuroscience

Activation of TGF/Smad signaling reduces aggregate formation of
mislocalized TDP-43
Satoshi Kaneko (Neurology)
Background: TAR DNA binding protein of 43 kDa (TDP-43) is naturally located in the nucleus
and has been identified as the major component of cytoplasmic ubiquitinated inclusions in patientswith amyotrophic lateral sclerosis (ALS). We have reported that TDP-43 and phosphorylated Smad2(pSmad2), an intracellular mediator protein of Transforming Growth Factor-(TGF) signaling, areco-localized within cytoplasmic inclusions in the anterior horn cells of sporadic ALS patients.
Objective: To investigate the possible pathophysiological linkage between pathologic cytoplasmic
inclusions containing TDP-43 and TGF/Smad signaling.
Methods: We replicated cytoplasmic aggregates of TDP-43 in HEK293T cells by transfecting thecells with a nuclear localization signal deletion mutant of TDP-43 and inhibiting proteasome activity,-55-

and assessed the effect of TGF/Smad signaling on the cytoplasmic aggregate formation.
Results: The aggregates contained ubiquitinated, phosphorylated, and fragmented TDP-43,
consistent with the essential features of the human pathology. Moreover, the aggregates wereco-localized with pSmad2 under continuous TGFstimulation. Overexpression of Smad2 reduced
the amount of cytoplasmic aggregates in HEK293T cells, and TGFstimulation augmented thisreduction effect in a dose-dependent manner.
Conclusion: Activation of TGF/Smad signaling system is protective against aggregate formation ofcytoplasmically mislocalized TDP-43 and may be a potential therapeutic approach to delay
progression of ALS.
1. Nakamura M, Kaneko S, Ito H, Jiang S, Fujita K, Wate R, Nakano S, Fujisawa JI, Kusaka H. Activation ofTransforming Growth Factor-β/Smad Signaling Reduces Aggregate Formation of Mislocalized TAR
DNA-Binding Protein-43. Neurodegener Dis. 11, 182-193, 2012.
2. Nakamura M, Kaneko S, Wate R, Asayama S, Nakamura Y, Fujita K, Ito H, Kusaka H. Regionally different
immunoreactivity for Smurf2 and pSmad2/3 in TDP-43-positive inclusions of amyotrophic lateral sclerosis.
Neuropathol. Appl. Neurobiol. in press, 2012.

Two separate neural systems for understanding other’s actions
Masaki Isoda (Cognitive Neuroscience)
Understanding the action of other individuals is of critical importance in the social world as itallows for a grasping of their mental states, such as beliefs, desires, and intentions. It has been
proposed that the brain has two separate systems for such action understanding –– the mirror systemand the mentalizing system. The mirror system mainly consists of the ventral premotor cortex andinferior parietal lobule and is hypothesized to play a role in understanding what others are doing via
embodied simulation. The mentalizing system, on the other hand, is mainly composed of the medial
frontal cortex and temporoparietal junction and assumed to play a part in understanding why others
are doing via mental simulation. These speculative roles are advocated on the basis of macro-level
analyses of human neuroimaging data. Accordingly, it remains unclear whether and how response
properties of individual neurons differ between the two systems during action observation.
Answering this question requires use of experimental animals and methodologies with a muchhigher spatiotemporal resolution. In this talk, I will review single-neuron recording data obtained in
the monkey brain, showing that mirror neurons nondifferentially respond to the actions of one’s own
and those of others, whereas mentalizing neurons can selectively respond to each of them. Thus, it
seems likely that the brain is equipped with dual systems that complement each other forunderstanding others: the mirror system whereby one can view others as analogous to oneself andthe mentalizing system whereby one can identify them as unique.
1.
2.

Yoshida, K., Saito, N., Iriki, A. and Isoda, M. Social error monitoring in macaque frontal cortex. Nat. Neurosci.
15, 1307-1312, 2012.
Yoshida, K., Saito, N., Iriki, A. and Isoda, M. Representation of others’ action by neurons in monkey medial
frontal cortex. Curr. Biol. 21, 249-253, 2011.

Visualization of “hypoxia” in vivo: imaging of HIF-1 activation
Kiichi Hirota (Anesthesiology)
For all organisms, changes in oxygen (O2) concentration represent a fundamental physiologic
stimulus. Particularly in vertebrate animals, either deficiency or excess of O2 elicits acute(rapid-onset and short-term) and chronic (delayed- onset and long-term) responses.
Oxygen plays a critical role in cellular homeostasis and many human diseases, including ischemic
diseases and cancer, are characterized by inadequate tissue oxygenation. In the former, lack of-56-

oxygen contributes to cell death and, in the latter, lack of oxygen is an early signature of disease thatmight also affect malignant cell behavior. Oxygen deprivation in human tissue leads to the
up-regulation of a complex array of genes that exert adaptive functions, particularly with regard tocellular metabolism and improvement in O2 delivery.
One of the most important transcription factors controlling O2-tension regulated gene expression
is hypoxia-inducible factor 1 (HIF-1). HIF-1 is a heterodimer containing the constitutively expressed
HIF-1β subunit and the inducible HIF-1α subunit, which binds the consensus sequence hypoxia
responsive element (HRE) in the expression regulatory region. Regulation of HIF-1 activity occurs
at multiple levels in cells, and mechanisms regulating HIF-1α protein expression and transcriptional
activity have been extensively analyzed. The von Hippel–Lindau tumor suppressor protein has been
identified as a HIF-1α-binding component of an ubiquitin–protein ligase that targets HIF-1α forproteasomal degradation in non-hypoxic cells. Under hypoxic conditions, hydroxylation of
HIF-1-specific proline and asparagine residues is inhibited due to substrate (O2) limitation, resulting
in HIF-1α protein stabilization and transcriptional activation.
This time, I will talk about the transgenic mice that carry HRE/ODD(oxygen-dependentdegradation domain)-luciferase (HOL) gene that generates bioluminescence in an HIF-1-dependent
manner and its successful usage to monitor HIF-1 activity in ischemic tissues and postnatal hypoxia.
1.
2.

Harada H et al. Cancer cells that survive radiation therapy acquire HIF-1 activity andtranslocate towards tumour
blood vessels. Nat. Commun. 3, 783, 2012.
Kadonosono T, et al. Detection of the onset of ischemia andcarcinogenesis by hypoxia-inducible transcription
factor-based in vivo bioluminescence imaging. PLoS ONE, 6, e26640, 2011.

Molecular Imaging for Diseases Using Mass Spectrometry
Ikuko Yao (Molecular and Functional Biology)
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) provides themeans for analysis of a wide variety of biomolecules. The technology, a combination of MALDI-MSand microscopy which is known as imaging mass spectrometry (IMS), provides us a novel
opportunity to visualize molecules directly on tissue surfaces without the need for target-specificreagents such as antibodies. As most of previous IMS studies analyzed the relative abundances ofmetabolite species, it is important to expand its application to various biomolecules for exploring
molecular basis of diseases. Here, we will show some examples of IMS application to diverse typesof biomolecules.
We applied IMS technique to directly detect neurotransmitters in the central nervous system(CNS) and visualize their localization in tissue samples. In MS/MS imaging, the transmitter could be
visualized with a high signal/noise ratio (S/N) by elimination of matrix interference. In the tandem
MS-imaging analysis of ACh in the mouse spinal cord, reconstructed ion intensity maps for ACh ion
showed that the ion which corresponds to ACh distributed around large motor neurons in the spinal
ventral horn. Similarly, we analyzed mouse brain sections with tandem MS imaging. In comparison
with the previous reports, it was revealed that ACh was localized in the region abundant inacetylcholinestelase. These results show that ACh is the suitable molecular species to detect inpositive ion detection mode by IMS, and that tandem MS imaging is useful for the analyses ofcholinergic neurons in the postmortem brain of neurodegenerative patients.
We also applied the IMS technique to formalin-fixed paraffin-embedded (FFPE) samples to
identify a substance(s) responsible for the intestinal obstruction caused by an unidentified foreign
body. In advance of IMS analysis, some pre-treatments were applied. After deparaffinization ofsections, samples were subjected to enzyme digestion. The sections co-crystallized with matrix weredesorbed and ionized by a laser pulse with scanning. A combination of enzyme digestion and the
2,5-dihydroxybenzoic acid (DHB) matrix gave best mass spectrum. These results for the first timeshow that MALDI-IMS is an accurate technique with high sensitivity for direct analysis of-57-

oligosaccharides in histopathological samples.
Our study shows that the direct detection of biomolecules will be useful for assessments such as
pathogenesis and evaluations of therapeutic strategies.
1.
2.

Sugiura Y, Zaima N, Setou M, Ito S, Yao I. Visualization of acetylcholine distribution in central nervous systemtissue sections by tandem imaging mass spectrometry. Anal. Bioanal. Chem. 403, 1851-1861, 2012.
Yamada M, Yao I, Hayasaka T, Ushijima M, Matsuura M, Takada H, Shikata, N, Setou M, Kwon AH, Ito S.
Identification of oligosaccharides from histopathological sections by MALDI imaging mass spectrometry. Anal
Bioanal Chem. 402, 1921-1930, 2012.

Session 3

Division of Cancer

Clinical applications of indocyanine green-fluorescence imaging to liver
surgery
Morihiko Ishizaki (Surgery)
BACKGROUND: Recently, some studies have reported the usefulness of intraoperative fluorescentimaging using indocyanine green (ICG) for the detection of sentinel nodes in breast and gastriccancer. These techniques are based on the finding that ICG binds to plasma proteins andprotein-bound ICG emits near-infrared light. Fluorescent imaging using ICG also has the potential todetect liver cancers through the visualization of the disordered biliary excretion of ICG in livercancer tissues compressed by the tumor, and to preventing postoperative bile leakage by using ICG
fluorescent cholangiography.
METHODS: Intravenous injection of the ICG reagent was performed as a liver function test 1 to 2
weeks prior to surgery. ICG fluorescent imaging was performed using the Photodynamic Eye (PDE)
infrared camera (Hamamatsu Photonics k. k., Shizuoka, Japan). 102 patients who underwent hepatic
resection without biliary reconstruction were divided into 2 groups. The control group (n = 50)
underwent a leak test with ICG dye alone, and the experimental group underwent a leak test withICG dye, followed by ICG fluorescent cholangiography using the PDE (PDE group, n = 52).
RESULTS: ICG fluorescent imaging was useful for liver surgical navigation as follows: three casesof recurrent hepatocellular carcinoma(HCC) after transcatheter arterial chemoembolization or
thermal ablation therapy; three cases of colorectal metastatic liver tumors after chemotherapy as
conversion therapy; two cases of intrahepatic cholangiocellular carcinoma (ICC); and two cases of
surface HCC that were not detected by preoperative computed tomography (CT) scan. In the ICC
cases, although dilatation of the bile duct could be detected, the tumor margins were ambiguous by
ultrasonography(US). ICG fluorescent imaging clearly described the dilatation of the bile duct
including the tumor. Among 42 patients with fluorescence in the PDE group, 25 patients had
insufficient closure of bile ducts on the cut surface of the liver, which were closed by suture or
ligation. There were 5 patients who developed postoperative bile leakage in the control group versus
no bile leakage in the PDE group (10% vs 0%, P = .019).
CONCLUSIONS: ICG fluorescent imaging was useful for liver surgical navigation including
selection of the mode of hepatic resection. ICG fluorescent cholangiography may have usefulpotential for prevention of bile leakage after hepatic resection.
1. Ishizawa, T. et al. Real-Time Identification of liver Cancers by Using ICG Fluorescent Imaging. Cancer, 115,2491-2504, 2009.
2. Kitai T, et al. Fluorescence navigation with ICG for detecting sentinel lymph nodes in breast cancer. Breast
Cancer, 12, 211-215, 2005.

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Mst1 regulates integrin-dependent thymocyte trafficking and antigen
recognition in the thymus
Yoshihiro Ueda (Molecular Genetics, IBS)
Thymocyte trafficking has an important role in thymic selection. Here we show that the Hippo
homologue Mst1 is required for thymocyte migration and antigen recognition by LFA-1 andICAM-1 within the medulla. Using two-photon imaging of thymic tissues, we found that highly
motile mature thymocytes arrest and are activated in the vicinity of rare populations of Aire(+)
ICAM-1(hi) medullary thymic epithelia in a negatively selecting environment. Notably, Mst1
deficiency or blocking the cell adhesion molecules LFA-1 and ICAM-1 results in inefficientmigration and antigen recognition of CD4(+) thymocytes within the medulla. Consistent with these
defects, thymocyte selection is impaired in Mst1(-/-) mice, which display T cell-dependent
inflammatory infiltrates in multiple organs and develop autoantibodies. Our results suggest that Mst1
has a key role in regulating thymocyte self-antigen recognition in the medulla.
1.
2.

Ueda Y, Katagiri K, Tomiyama T, Yasuda K, Habiro K, Katakai T, Ikehara S, Matsumoto M, Kinashi T. Mst1
regulates integrin-dependent thymocyte trafficking and antigen recognition in the thymus. Nat. Commun. 3,1098, 2012.
Katagiri K, Katakai T, Ebisuno Y, Ueda Y, Okada T, Kinashi T. Mst1 controls lymphocyte trafficking andinterstitial motility within lymph nodes. EMBO J. 28, 1319-1331, 2009.

Development of a highly efficient method for isolating bone-derived small
stem cells identified in adult mouse bone
Ryusuke Nakatsuka (Stem Cell Biology)(Background) Ratajczak and his colleagues identified a unique population of very small
embryonic-like (VSEL) stem cells in adult mouse bone marrow (BM) (Leukemia 2006:20;857).
However, it is difficult to isolate these small stem cells (SSC) in BM very effectively. This study
describes our recently developed highly efficient method for isolating SSCs using enzymatic
treatment of murine bone.
(Materials and Methods) The bone tissue from 8 week-old C57BL/6 mice were crushed in a mortar
and then incubated in cell dissociation buffer. Next, the BM nucleated cells (BMNC) andbone-derived nucleated cells (BDNCs) were stained with various monoclonal antibodies and thenwere used for subsequent FACS analyses.
(Results) The incidences of SSCs in the BMNCs and BDNCs were 0.001% and 0.1%, respectively.
Therefore, the enzymatic treatment of bone tissues yielded about 100 times the efficiency for theisolation of SSCs. The bone-derived (BD) SSCs were small and possessed a relatively large nucleus
surrounded by a narrow rim of cytoplasm. However, the gene expression profile of the BD-SSCs
was clearly distinct from the well-defined populations of ES cells, KSL cells, and MSCs. The
number of these BD-SSCs significantly increased after the induction of liver injury by carbon
tetrachloride administration. They were then most likely mobilized into the peripheral blood.
(Conclusion) The present data suggest that the majority of the Lin-Sca-1+CD45- cells reside in thebone tissue. The gene expression profile of BD-SSCs was different from those of the previously
reported BM-derived SSCs (VSELs) while BD-SSCs resemble BM-derived VSELs.
1.
2.

Bone marrow as a home of heterogenous populations of nonhematopoietic stem cells. Leukemia, 19, 1118-1127,
2005.
A population of very small embryonic-like (VSEL) CXCR4+SSEA-1+Oct-4+ stem cells identified in adult bonemarrow Leukemia, 20, 857-869, 2006.

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Closing remarks
Hiroo Ueno
Stem Cell Pathology
Generation of chimeras, which is now a standard technology for producing gene modified mutant
mice, was originally developed as a tool for developmental biology. However, application of
conventional single marker chimeric mice for developmental study had been limited. This situation
has been dramatically changed by development of multicolor chimeric mice using various kinds offluorescent proteins. Now by our technology, up to ten different clones could be distinguished in situ
by their colors, which enable us to perform more accurate statistical analyses and lineage tracing
experiments than conventional methods. The method could be applied to visualize not only cell
turnover of normal stem cells but also cancer development in vivo. As closing remarks, we will
briefly discuss what questions these methods are now answering and introduce lingual epithelial
stem cells we recently identified and reported.

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Research Activities 2011
平成 23 年度研究成果報告

Division of Neuroscience

神経部門

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Annual Report 研究成果報告書
In situ imaging of neural cells and their function
神経細胞機能の in situ イメージング

Title of Project
P.I.

Hisao Yamada, 山田久夫

E-mailyamada@takii.kmu.ac.jp

Affiliation

Regenerative Neural Medicine, 神経機能再生医学（解剖学第 1）講座

Collaborators

若林毅俊、森徹自、高森康晴、平原幸恵、小池太郎

Keywords

brain, glia, lamin, neuron, rat, retina, stem cell

1. SUMMARY OF RESEARCH RESULTS
Lamins are intermediate filament proteins that form nuclear lamina, which are divided into A/C-,
B1- and B2-isotypes.

Lamins are thought to play important roles in normal morphogenesis andcell-differentiation.

In the nervous system, their abnormality causes neurodegenerative diseases

such as peripheral neuropathy, leukodystrophy, lissencephaly etc.

We have been

immunohistochemically investigating the expression patterns of lamin isotypes in the adult rat
neural tissues.

In the neurogenic regions of adult rat brain, GFAP-positive neural stem cells

showed lamin A⁄C (++), B1 (++) and B2 (++), PSA-NCAM-positive progenitor cells showedlamin A⁄C (–), B1 (+++) and B2 (+), and mature neurons showed lamin A⁄C(++), B1 (+) and B2(+++), while mature oligodendrocytes and astrocytes showed lamin B2 (+).
retina, most of retinal neurons also expressed lamin B1, B2 and C.

In the adult rat

However, horizontal cells anda subpopulation of retinal ganglion cells also possessed lamin A; and photoreceptor cells
possessed neither lamin A nor C. The neural cells in the brain and retina dominantly expressedlamin C protein among the lamin A/C gene products,.

These results suggest that lamins are

involved in cell-differentiation and expression of cell-specific genes in individual neural cells.
核膜ラミナに存在するラミン（Lamin）というタンパク質は、選択的スプライシングによる A および C、異なる遺伝子に由来する B1 および B2 と言ったイソタイプをもつ。これらの遺伝子の変異は、早老症、成人発症型白質ジストロフィーをはじめとする神経筋疾患でみられる。このような疾患に関わる lamin または核ラミナをイメージング法でとらえ、そのイソタイプごとの分布を解析する研究を展開している。正常成獣ラットおよびマウスの海馬歯状回や側脳室周囲帯では、神経系幹細胞が A/C タイプおよび B1/B2 タイプに陽性で、やや分化した神経前駆細胞では A/C タイプが陰性になり B1 タイプが増加し B2 タ
イプが減少するが、成熟ニューロンでは A/C タイプが再び陽性で、B1 タイプと B2 タイプの密度が逆転し、B2 タイプ優位になることを報告した。一方、オリゴデンドロサイト
やアストロサイトと言った成熟マクログリアでは B2 タイプの濃度がニューロンよりやや薄いことなどを明らかにした。さらに、網膜の成熟ニューロンでは B1 タイプと B2 タイプがほぼ同等に分布すること、それに加えて、水平細胞やごく一部の神経節細胞では、A/C タイプも含有し、視細胞には A/C のどちらのタイプも存在しない事も証明した。脳や網膜の神経組織では laminA/C 遺伝子産物のうち、C タイプ優位であることが、他の組織と異なる。これらを総合すると、ラミンは細胞種特異的にそのイソタイプ構成を変化させながら、各細胞の分化に関わっていくと考えられる。
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2. List of Publications
Original Articles
1. Wakabayashi T, Mori T, Hirahara Y, Koike T, Kubota Y, Takamori Y, Yamada H
Nuclear lamins are differentially expressed in retinal neurons of the adult rat retina.
Histochem Cell Biol, 136(4):427-436, 2011.8.
Proceedings
1. 若林毅俊, 森徹自, 平原幸恵, 小池太郎, 高森康晴, 山田久夫
成獣ラットの網膜ニューロンにおけるラミンの発現パターン
第 117 回日本解剖学会総会・全国学術集会, 甲府, 2012.3
2. 紅林秀治, 森徹自, 若林毅俊, 山田久夫
海馬歯状回における神経細胞の増殖に影響を与える生育環境―『豊かな環境』を定量化した解析―
第 117 回日本解剖学会総会・全国学術集会, 甲府, 2012.3
3. Mori T, Wakabayashi T, Hirahara Y, Takamori Y, Koike T, Kurokawa K, Yamada H
The effects of seizures induced by pentylenetetrazole on cell cycle progression of adultsubependymal zone precursors.
Society for Neuroscience 41th Annual Meeting, Washington D.C., USA, 2011.11
4. 高森康晴, 森徹自, 若林毅俊, 平原幸恵, 小池太郎, 山田久夫
成獣ラットの脳の各種グリア細胞におけるラミン･サブタイプ構成の多様性
第 34 回日本神経科学大会, 横浜, 2011.9
5. 森徹自, 若林毅俊, 平原幸恵, 高森康晴, 小池太郎, 黒川清, 山田久夫
てんかん発作が成獣脳室下帯の神経前駆細胞に与える影響
第 34 回日本神経科学大会, 横浜, 2011.9-63-Annual Report 研究成果報告書
Title of Project

Striatal neurons highly expressing GAD1 mRNA are populated in the lateral
striatum
マウス線条体における calbindin-poor 領域の空間配置と GABA ニューロン

P.I.

Tetsuo Sugimoto, 杉本哲夫

Affiliation

Anatomy and Brain Science, 脳構築学（解剖学第 2）講座

Collaborators

Trifonov Stefan、山下雄司、宝谷剛志、丸山正人、加瀬政彦、清水順一

Keywords

calbindin, GABA neuron, lateral striatum

E-mailsugimoto@takii.kmu.ac.jp

1. SUMMARY OF RESEARCH RESULTS
GAD1 and GAD2 are universally contained in GABAergic neurons in the CNS. The two
isoforms are almost identically expressed throughout the brain and spinal cord. By using in situ
hybridization, we found that the mouse lateral striatum concentrates medium-sized projection
neurons with high-level expression of GAD1, but not of GAD2, mRNA. These findings wereconfirmed with different types of riboprobe including those directed to 5'-noncoding region, 3'noncoding region and coding region from mouse or rat genes. Immunohistochemical localizationof GAD1 also revealed the predominant localization of the enzyme in the same striatal region. The
lateral region of the mouse striatum, harboring such neurons, is ovoid in shape and extends
between interaural +4.8 and +2.8, and at lateral 2.8 and dorsoventral 2.0. This intriguing region
corresponds to the area that receives afferent inputs from the primary motor and sensory cortex
that are presumably related to mouth and forelimb representations. The lateral striatum is includedin the basal ganglia-thalamocortical loop and is most vulnerable to various noxious stimuli in theneurodegeneration processes involving the basal ganglia. The GAD1 mRNA distribution in themouse lateral striatum partially resembled those of GPR155 and cannabinoid receptor type 1
mRNAs, suggesting functional cooperation in some neurons. Next, it was examined by calbindin
immunostaining whether this intriguing region corresponds to the area fully included in thecalbindin-poor zones. The calbindin-poor zones were seen laterally and dorsolaterally in the
striatum, while predominant calbindin-rich matrix structures containing calbindin-poor patches
were seen in the central and ventromedial parts of the striatum. We found that the locations of cellgroups characterized by a high level of GAD1 mRNA correspond to the part of the lateral striatum
characterized as calbindin-poor zones.
線条体の外側部は感覚運動皮質から強い投射を受ける。この領域は、Calbindin D-28K
免疫染色で出現する Calbindin-poor compartment にほぼ一致することが知られている。しかしながらこれまで Calbindin-poor compartment の organization は判然としていない。一方、われわれの研究を含むこれまでの研究から、線条体の外側部に比較的強く発現するマーカー物質が知られている。本研究では、マーカー物質として、外側部
ニューロンに強く発現している GAD1 mRNA と Cannabinoid type 1 receptor (CB1)mRNA を用いて、それぞれの分布域をマウス線条体 Calbindin-poor compartment 内に特定した。その結果、(1) Calbindin-poor compartment は、大きく Anterior, Middle,
Posterior の３つの領域に区分される。(2) GAD1 mRNA は Middle region にとくに強く発現する。(3) CB1 mRNA は３つの領域に広く発現する。(4) Anterior region とPosterior region は、線条体の腹側部で合流する。背側部で拡散する。全体として、Middle
region をなかに挟んだ V 字型を描く。以上の (1) - (4) が判明した。Compartment の
このユニークな形態は、おそらく背腹方向に分布する同一の皮質対応部位の情報処理に関し、背側部と腹側部で機能が相当異なっていることを形態から示唆しているものと考
えられた。
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2. List of Publications
Original Articles
1. Stefan Trifonov, Takeshi Houtani, Masahiko Kase, Kazunori Toida, Masato Maruyama, Yuji
Yamashita, Jun-ichi Shimizu and Tetsuo Sugimoto. Lateral regions of the rodent striatum
reveal elevated glutamate decarboxylase 1 mRNA expression in medium-sized projection
neurons. Eur. J. Neurosci., 35 (5): 711-722, Feb. 15, 2012.
2. Satoko Hamada, Stefan Trifonov, Takeshi Houtani, Masahiko Kase, Yuji Yamashita, Kazuyasu
Baba, Jun-ichi Shimizu, Masato Maruyama, Koichi Tomoda, Toshio Yamashita and Tetsuo
Sugimoto. Cholinergic Fibers and Muscarinic Receptors in the Central Auditory System. J.
Kansai Med. Univ., in press, 2012.
Proceedings
1. 山下雄司，Stefan Trifonov，宝谷剛志，丸山正人，加瀬政彦，杉本哲夫．マウス線条体
外側部マーカー物質の V 字型縞構造への局在．第 117 回日本解剖学会・全国学術集会，甲府，2012 年 3 月．
2. 山下雄司，Stefan Trifonov，宝谷剛志，丸山正人，加瀬政彦，杉本哲夫．マウス線条体の V 字型構造．第 87 回日本解剖学会近畿支部学術集会，西宮，2011 年 12 月．
3. 山下雄司，Stefan Trifonov，宝谷剛志，丸山正人，加瀬政彦，清水順一，杉本哲夫．マ
ウス線条体外側部 calbindin-poor 領域の空間配置．第 34 回日本神経科学大会，横浜，
2011 年 9 月．

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Annual Report 研究成果報告書
Title of Project

Imaging of disease-related molecules and cells
病因細胞および分子のイメージング

P.I.

Hiroko Matsuda, 松田博子

E-mailmatsudah@takii.kmu.ac.jp

Affiliation

Cellular and Molecular Physiology, 細胞分子生理学（生理学第 1）講座

Collaborator

松田博子、岡田誠剛、武藤恵、林三樹夫

Keywords

ion channel, proteolysis, virus vector, visualization

1. SUMMARY OF RESEARCH RESULTS
Protein expression is regulated by synthesis as well as degradation, but little is known
about the degradation of inwardly rectifying K+ channel (Kir2.1). We developed two in vivo
applicable methods for the examination of protein degradation using fluorescent proteins, i.e.,
SNAP-tag and fluorescent timer (FT). We constructed the SNAP-Kir2.1 and FT-Kir2.1 fusion
proteins and revealed that the degradation of Kir2.1 is regulated through the current.
Pulse-chase experiments showed that the half-life of SNAP-Kir2.1 is shortened in highexpression cells, the blockade of Kir current elongated the half-life, and the half-life of
hyper-conductive mutant Kir2.1 is shorter than that of wild-type. The fluorescence of FTchanges spontaneously and slowly from green to red; thereby the green/red ratio allows us
detect the changes in the half-life of the FT-fused protein. We constructed of FT-Kir2.1 fusion
protein and confirmed the slowed degradation by the current blockade.
High titer of viral vector is essential for the efficiency of the study. We found that the titerof the lentiviral vector, which expresses Kir2.1, is increased by the blockade of Kir2.1 current
during viral production. Reportedly, cellular ATP-level is critical for the viral vector
production, therefore we measured the ATP level of the viral vector producing cells.
Expectedly, the ATP level was decreased by the overexpression of Kir2.1, and the decrease
was restored by the blockade. Interestingly, the blockade did not increase the titer of otherlentiviral vectors that express voltage-dependent K+ channels, i.e., Kv1.4, Kv4.2, and HERG.
These results suggest the increase in the titer is dependent on the voltage-dependency of theK+ channels.
Conventional assay for ion channels is time-consuming and requires the skill ofpatch-clamping or costly patch-clamping robot. We developed a labor-saving assay for Kir2.1,
based on the toxic effect of excessive K+ current and coexpressed GFP fluorescence. Thismethod enables us high-through-put assays for blocker and opener of Kir2.1. Using thismethod, we started the screening of the venom of Mexican scorpions, which is a promisingsource for the blocker and opener of ion channels.
To construct a viral vector which specifically expresses a gene in somatotrophs, weconstructed lentiviral vectors, which express GFP under the control of promoter of growth
hormone gene, and injected into the rat pituitary. The lentiviral vector robustly expressed the
GFP in a somatotroph-specific way. The expression of GFP in upregulated by GHRH and a
IGF-1 receptor blocker, showing the retention of transcriptional control. Furthermore, theexpression of GFP was reduced by mental stress, which is reported to reduce the human
growth. Next, for the examination of the development of somatotroph, we constructed the
Moloney retroviral vector of similar construct. The retroviral vector expressed GFP in the
newborn somatotrophs. The GFP-positive cell tended to form clusters. Our cluster analyses-66-suggest preceding determination of somatotrophs and delayed expression of growth hormone.
GFP was transiently expressed by lactotrophs, suggesting a similarity in the transcriptional
control.
To examine the ion channels involved in the secretion of HCO3- from the duct cell in thepancreas, we established the monolayer and oriented culture of Capan-1 cells on a membranefilter. This oriented culture recapitulates the apical and basolateral structures of the duct. We
found the apical distribution of KCa3.1, Kv7.1, CFTR, Adenosine2A receptor, and thebasolateral expression of KCa3.1 and secretin receptor. We confirmed the similar expression
pattern in the mouse pancreas. Secondly, to express a gene of interest in a duct cell-specificway, we constructed a lentiviral vector that express GFP under the control of secretin receptor
gene. This lentiviral vector successfully expressed the GFP in the Capan-1cells.
① 特定のタンパクの分解速度の変化を、蛍光タンパクを用いた方法を開発し、それを用いて内向き整流性 K+チャネル(Kir2.1)の分解が K+電流に応じて調節されることを明らかにした。
② Kir2.1 を発現するレンチウイルスベクターの調製時に、同チャネル電流を遮断すると、タイターが上昇することが見出した。
③ 過剰な K+電流による毒性と、共発現させた GFP 蛍光を用いて、Kir2.1 電流のHigh-through-put なアッセイ系を開発した。これを用いて、これまでに他のイオンチ
ャネル遮断薬、開口薬が発見されているメキシコ産サソリ毒のスクリーニングを開始した。
④ 成熟および新生ソマトトロフをラベルし、ストレスに対する反応、および分化を検討するために、成長ホルモン(GH)プロモーターを用いたレンチ及びレトロウイルス
ベクターを作成し、ソマトトロフ特異的なレポーター遺伝子(GFP)の発現を認めた。
GFP 発現はストレスによって低下した。
⑤ 膵導管細胞の HCO3-分泌に関与するイオンチャネルを明らかにするため、１）膵癌
Capan-1 細胞をフィルター上で単層培養し、上皮細胞モデルを作製した。KCa3.1,
Kv7.1, CFTR、アデノシン 2A 受容体の管腔側の発現、セクレチン受容体の側底膜側の発現を認めた。マウス膵導管において、同様の発現パターンを認めた。2)導管細胞
特異的な遺伝子発現を可能にするため、セクレチン受容体プロモーターを用いたレンチウイルスベクターを作成し、Capan-1 細胞でレポーター遺伝子(GFP)の発現を認めた。

2. List of Publications
Proceedings
1. 林美樹夫、ノバック・イヴァナ・マウス膵導管微小灌流標本における静止膜電位・日本生理学会、松本・2012 年 3 月 30 日
2. 武藤恵、松田博子・海馬自発性電流の修飾・日本生理学会・松本・2012 年 3 月 29 日

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Annual Report 研究成果報告書
Title of Project

The role of serotonin and dopamine for cognitive processes in the primate.
霊長類におけるセロトニン・ドパミンによる認知機能の制御

P.I.

Kae Nakamura, 中村加枝

E-mailnakamkae@takii.kmu.ac.jp

Affiliation

Cognitive Neuroscience, 高次認知脳科学（生理学第 2）講座

Collaborators

松崎竜一、則武厚、林和子

Keywords

dopamine, dorsal raphe nucleus, hypothalamus, monkey, reward, serotonin

1. SUMMARY OF RESEARCH RESULTS
Decision making based on reward and punishment is critical for animals’ survival. While many
brain areas have been implicated in the process, exactly which area is computing which aspect of
reward or punishment has not been well understood. To this end, we recorded single neuronal
activity of neurons in the dorsal raphe nucleus (DRN), the major source of serotonin, and thelateral hypothalamic area (LHA), one of the main source of dopamine neuronal activity which
encodes reward predication error, while monkeys performed (1) saccade tasks with biased reward
schedule and (2) Pavlovian conditioning task.
In the DRN, we found that many neurons responded stronger either for rewards andpunishments. However, the response was markedly stronger for the tasks with actions (i.e. saccade
tasks) rather than the passive task (i.e. Pavlovian conditioning task). Thus the DRN appeared to be
involved in integration of value and action.
In the LHA, we found many neurons showed modulation in activity depending on the reward
predictability. Some neurons also showed stronger response to aversive events. Thus the LHA
carries enough information to compute positive and negative value. We will further investigate
how these areas communicate each other commnuicate to integrate the information, by
pharmacological and electrophysiological approaches.
報酬とそれを得るためのコスト・嫌悪刺激の情報を計算して行動を選択することは、生物の生存に決定的な影響を与える。報酬や嫌悪刺激の情報処理にかかわる脳領域は多く報告されているが、どの領域が報酬情報の中でも具体的に何を計算しているのか、情報がどのように統合されて最終的な行動に至るのかいまだによくわかっていない。この疑問に答えるためには、同じ個体、同じ行動課題で、異なる領域から神経活動を記録する方法が有効である。そこで我々は、カニクイザル計 3 頭において、報酬量を変化させた眼球運動課
題と、パブロフ型の条件付け課題、さらに go nogo 課題を訓練し、セロトニン細胞の存
在する背側縫線核（DRN）、ドパミン系に強い投射がある視床下部外側部（LHA）から単一神経細胞外記録を行った。それぞれの領域では異なる形で報酬や嫌悪刺激情報が表現されていることがあきらかになった。DRN：パブロフ型の条件付け課題において、報酬に対しては予測される報酬よりも、予測されない報酬に対して、しばしば強く応答した。嫌悪刺激に対しては、一般に反応は弱かった。これらの結果は、DRN 細胞はドパミン神経と同様に報酬予測において重要な役割を担う可能性を示唆する。LHA：従来は正の報酬に応答すると考えられていたが、正のみならず負の報酬に応答するものがあり、さらに報酬
確率をコードしている細胞群も見つかった。これらのことは、ＬＨＡが正負報酬の統合処理を行うに十分な情報量をもっていることを意味する。
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2. List of Publications
Original Articles
1. Okada K, Nakamura K, Kobayashi Y・A neural correlate of predicted and actual reward-value
information in monkey pedunculopontine tegmental and dorsal raphe nucleus during saccade
tasks.・Neural Plasticity・Volume 2011, Article ID 579840・2011・1-21
Proceedings
1. Atsushi Noritake, Kae Nakamura・Neuronal modulation in appetitive and aversive contexts inthe primate lateral hypothalamus・日本神経科学大会・パシフィコ横浜（神奈川）・2011
年9月
2. Kazuko Hayashi, Atsushi Noritake, Kae Nakamura・Cognitive control of response inhibi
tion in the primate・日本神経科学大会・パシフィコ横浜（神奈川）・2011年9月
3. Kae Nakamura・APPETITIVE AND AVERSIVE CODING BY THE PRIMATE DORSAL RAPHE NUCLEUS・11th International Conference on Cognitive Neuroscience・Mallorca , Spain・2011年9月
4. Atsushi Noritake, Kae Nakamura・Comparison of neuronal signals for reward value inappetitive and aversive contexts in the primate lateral hypothalamus area・Society for
Neuroscience Meeting・Washington, DC, USA・2011 年 11 月
5. Kazuko Hayashi, Kazuko Nakao, Ryuichi Matsuzaki, Ken-ichi Okada, Yasushi Kobayashi,
Kae Nakamura・Neuronal activity in the primate dorsal raphe nucleus encodes positive andnegative value・Society for Neuroscience Meeting・Washington, DC, USA・2011年11月-69-

Annual Report 研究成果報告書
Title of Project

Elucidation of pathogenesis and development of diagnosis and treatment of
intractable neural diseases
難治性神経疾患の病態解明と診断治療法の開発

P.I.

Seiji Ito, 伊藤誠二

E-mailito@takii.kmu.ac.jp

Affiliation

Molecular and Functional Biology, 分子生体機能学（医化学）講座

Collaborators

松村伸治、矢尾育子、下條正仁、片野泰代、西田和彦

Keywords

gene-engineered mice, imaging mass spectrometry, neurodegenerative diseases,
nocistatin, pain

1. SUMMARY OF RESEARCH RESULTS
These 10 years we prepared neuropathic pain model in many gene-engineered mice, and havebeen engaged in the elucidation of its pathogenesis and search of target molecules in the spinal
cord.

We found that neural plasticity mediated by glutamate receptors, especially NMDA

subtypes is involved in the generation and maintenance of chronic pain and that there is asimilarity in neural plasticity and molecules between central sensitization observed in the spinal
cord and long-term potentiation in the hippocampus. On the other hand、while candidates of
causative genes and proteins

are known in neurodegenerative disorders such as amyotrophic

lateral sclerosis and Huntington disease, their effective treatments have not been developed.

In

addition to conventional research with molecular, biochemical, and immunochemical procedures,
we study normal neuronal transmission in vivo and elucidation of pathogenesis and search oftarget molecules with state-of-the-art imaging systems, including by multi-photon microscopy andimaging mass spectrometry. Furthermore, we have started to prepare gene-engineered mice fortarget molecules searched by us and clarify a mechanism of diseases with patients’ iPS cells.
Major research activities in 2011 are as follows.
1) Elucidation of mechanism of inflammatory and neuropathic pain in model animals (N5-4, -8,-11)2) Identification of a target protein of nocistatin and confirmation by its knockout mice (N5-12)
3) Role of transcription factors in development and differentiation (N5-2, -10)
4) Synthesis of 11C-labeled kainoid for PET and its functional analysis（N5-6）
5) Development of software and application of imaging mass spectrometry（N5-1, -12, -15）
6) Development of novel strategy of the control of postoperative pain by analgesic hydrogel（N5-14）
この 10 年間、様々な遺伝子改変マウスで神経障害性疼痛モデルを作製し、その病態解明と標的分子の探索を脊髄で行ってきた。その結果、（１）グルタミン酸受容体の可塑的変化とその下流の分子が慢性疼痛に関与すること、（２）脊髄での神経可塑的変化は、海馬など上位中枢でみられる長期増強の可塑的変化と共通性があることがわかった。一方、筋萎縮性側索硬化症、ハンチントン病などの神経変性疾患では、その原因遺伝子・タンパ
ク質が見つかっているが、有効な診断治療法はない。従来の共焦点顕微鏡に加えて、PET、多光子励起顕微鏡、質量顕微鏡等の最新の分子イメージングシステムを用いて、正常な神経系での情報伝達機構とその病態の解明と標的分子の探索を行っている。探索した標的分-70-

子のノックアウトマウスを作製する一方、患者 iPS 細胞を用いた病態解明も進めている。
平成 23 年度の主な成果は以下のとおりである。
１）モデル動物を用いた炎症性、難治性疼痛の発症・維持機構の解明（N5-4, N5-8, N5-11）２）ノシスタチン標的分子 NIPSNAP1 の同定とノックアウトマウスでの検証 (N5-12)３）転写因子の神経発生・細胞分化の役割の解明（N5-2, N5-10）
４）PET 用 11C カイニン酸誘導体の合成と機能解析（N5-6）５）質量顕微鏡のソフトウェアの開発と応用（N5-1, N5-12, N5-15）６）生体吸収性ゲルの手術創埋め込みによる新しい術後鎮痛法の開発（N5-14）

2. List of Publications
Original Articles
1. Hayasaka, T., Goto-Inoue, N., Ushijima, M., Yao, I., Yuba-Kubo, A., Wakui, M., Kajihara, S.,
Matsuura, M. and Setou, M. Development of imaging mass spectrometry (IMS) dataset extract
software. Anal. Bioanal. Chem. 401, 183-193, 2011.
2.

Shimojo, M. RE1-silencing transcription factor (REST) and REST-interacting LIM domain
protein (RILP) affect P19CL6 differentiation. Genes Cells 16, 90-100, 2011.

3.

Ohnaka, M., Okuda-Ashitaka, E., Kaneko, S., Ando, A., Maeda, M., Furuta, K., Suzuki, M.,
Takahashi, K., and Ito, S. Induction of arginase II mRNA by nitric oxide using an in vitro
model of gyrate atrophy of choroid and retina. Invest. Ophthalmol. Vis. Sci. 52, 1493-1500,
2011.

4.

Katano, T., Nakazawa, T., Nakatsuka, T., Watanabe, M., Yamamoto, T. and Ito, S.
Involvement of spinal phosphorylation cascade of Tyr1472-NR2B, Thr286-CaMKII, andSer831-GluR1 in neuropathic pain. Neuropharmacol. 60, 609-616, 2011.

5.

Yao, I., Takao, K., Miyakawa, T., Ito, S. and Setou, M. Synaptic E3 Ligase SCRAPPER in
Contextual Fear Conditioning; Extensive Behavioral Phenotyping of Scrapper Heterozygote
and Overexpressing Mutant Mice. PLoS ONE 6, e17317, 2011.

6.

Kanazawa, M., Furuta, K., Doi, H., Mori, T., Minami, T. and Ito, S. Synthesis of an acromelicacid A analog-based 11C-labeled PET tracer for exploration of the site of action of acromelicacid A in allodynia induction. Bioorg. Med. Chem. Lett. 21, 2017-2020, 2011.

7.

Chizaki, R., Yao, I., Katano, T., Matsuda, K. and Ito, S. Restricted expression of
Ovol2/MOVO in XY body of mouse spermatocytes at the late pachytene stage. J. Androl. 33,
2, 2011.

8.

Lu, J., Katano, T., Okuda-Ashitaka, E., Oishi, Y., Urade, S. and Ito, S. Rapid S-nitrosylationof actin by NO-generating donors and in inflammatory pain model mice. Mol. Pain, 7, 101,
2011.

9.

Okazaki, T., Otani, H., Shimazu, T., Yoshioka, K., Fujita, M., Katano, T., Ito, S. and Iwasaka,
T. Reversal of inducible nitric oxide synthase uncoupling unmasks tolerance toischemia/reperfusion injury in the diabetic rat heart. J Mol Cell Cardiol. 50, 534-44, 2011.

10. Nishida, K., Nakayama, K., Yoshimura, S. and Murakami, F. Role of Neph2 in pontine nuclei
formation in the developing hindbrain. Mol. Cell. Neurosci. 46, 662-670. 2011.
11. Nishiyori, M., Uchida, H., Nagai, J., Araki, K., Mukae, T., Kishioka, S. and Ueda, H.
Permanent relief from intermittent cold stress-induced fibromyalgia-like abnormal pain byrepeated intrathecal administration of antidepressants. Mol Pain. 7, 69, 2011.
12. 伊藤誠二, 南敏明 （2011） 医薬品開発のための慢性疼痛発生・維持機構の考え方. 医-71-

薬品医療機器レギュラトリーサイエンス, 第42巻, 第５号, 392-399頁.
13. 南敏明、伊藤誠二（2011）医薬品開発のためのペインクリニックの現状
機器レギュラトリーサイエンス, 第 42 巻 第 6 号, 474-480 頁

医薬品医療14. 伊藤誠二、下條正仁、松村伸治（2011）痛みの可塑性と慢性化 脊椎脊髄ジャーナル
特集痛みとしびれのサイエンス─基礎と臨床, 第 24 巻 第 5 号, 341-347 頁
15. 芦高恵美子、伊藤誠二（2012）ノシセプチンとノシスタチン
刊臨床神経科学 2 月号, 第 30 巻 第 2 号, 148-151 頁

Clinical Neuroscience 月

Proceedings
1. Ito, S. Mechanism of maintenance of neuropathic pain in the spinal cord, as a model of neuralplasticity. The 6th International Conference of Neurons and Brain Diseases in Toyama,
Toyama, August 3, 2011.
2. Ito, S., Kunori, S., Matsumura, S., Katano, T., Urade, Y. and Okuda-Ashitaka, E. Blockade of
microglial migration in the spinal cord by prostaglandin E2 via EP2. The 41st Annual Meetingof the Society for Neuroscience, Washington, November 12-16, 2011.
3. Yao, I., Katano, T., Miyakawa, T., Ito, S and Setou, M. Synaptic E3ligase SCRAPPER is
crucial for hippocampus-dependent fear memory formation. The 41st Annual Meeting of theSociety for Neuroscience, Washington, November 12-16, 2011.
4. Katano, T, Ito, S. The activation of NR2B-NMDAR by phosphorylation of Tyr1472-NR2B
triggers phosphorylation cascade in neuropathic pain. The 41st Annual Meeting of the Society
for Neuroscience, Washington, November 12-16, 2011.
5. 向真樹、片岡達彦、大谷晃宏、久保幹、山田久夫、伊藤誠二、木原裕. 医薬理工融合型ライフサイエンス分野における高度人材育成教育プログラムの創成 第 43 回日本医学教育学会大会、ポスター発表、広島、2011、7 月
6. 荒木吉朗、海堀昌樹、松村伸治、伊藤誠二、權雅憲. 生体吸収性ハイドロゲルの手術
創埋込みによる鎮痛薬の徐放効果を用いた革新的な術後鎮痛法の開発 日本ペインク
リニック学会第 45 回大会、ポスター発表、松山、2011、7 月-72-

Annual Report 研究成果報告書
Title of Project

Analysis of intracytoplasmic inclusions of neurodegenerative diseases byimaging mass spectrometry
質量顕微鏡を用いた神経変性疾患細胞内封入体の研究

P.I.

Hirofmi Kusaka, 日下博文

E-mailkusaka@takii.kmu.ac.jp

Affiliation

Neurology, 神経内科学講座

Collaborators

金子鋭、和手麗香、藤田賢吾、朝山真哉

Keywords

amyotrophic lateral sclerosis, inclusion body, neurodegenerative disease,progressive supranuclear palsy

1. SUMMARY OF RESEARCH RESULTS
Before we start analysis of human brain section by imaging mass spectrometry, we performed
immunohistochemical studies of intacytoplasmic inclusions of neurodegenerative diseases toobtain further preliminary findings.1)
We investigated a family manifesting amyotrophic lateral sclerosis (ALS) with a heterozygous
E478G mutation in the optineurin (OPTN) gene. TAR DNA-binding protein 43 (TDP-43)-positive
neuronal intracytoplasmic inclusions were reactive with anti-ubiquitin and anti-p62 antibodies, but
negative for anti-OPTN antibody. Immunoreactivity of nuclear TDP-43 was reduced ininclusion-bearing cells. Fragmentation of Golgi apparatus was identified in 70% of the anterior
horn cells. The presence of anterior horn cells with preserved nuclear TDP-43 and a fragmented
Golgi apparatus indicates that patients with the E478G OPTN mutation would manifest
fragmentation of Golgi apparatus before loss of nuclear TDP-43.2)
Immunohistochemical analysis revealed that all the basophilic inclusions (BIs) were positive for
OPTN, Fused in sarcoma (FUS) and myosin VI in sporadic basophilic inclusion body disease(BIBD) patients and familial ALS with FUS mutation. However, the BIs showed no
immunoreactivity for TDP-43 or SOD1. Therefore, OPTN associates with each of the 3 major
ALS-related proteins, i.e., TDP-43, SOD1, and FUS. FUS is known to act as a co-activator of
NF-kB, and OPTN negatively regulates NF-kB activation. Therefore, sequestration of both FUS
and OPTN in BIs may induce dysregulation of NF-kB activation, leading to neurodegeneration.
Moreover, OPTN and myosin VI play a role in the maintenance of Golgi apparatus. Theirsequestration in BIs may be an underlying pathomechanism of fragmented Golgi apparatus.3).
We demonstrated for first time that Smad ubiquitination regulatory factor-2 (Smurf-2) isco-localized in the phosphor-tau inclusions in progressive supranuclear palsy (PSP). As Smurf-2 isan E3 ligase of Smad2, intracellular signaling component of TGFb, dysregulation of TGFb/Smadsignaling may be an underlying pathomechanism of PSP.4) Protein disulfide isomerase (PDI) is amember of the thioredoxin superfamily and is believed to accelerate the folding of
disulfide-bonded proteins by catalyzing the disulfide interchange reaction. We demonstrated that
PDI is co-localized in cytoplasmic inclusions of familial and sporadic ALS. Sequestration of PDI
may result in accumulation of misfolded proteins in ALS.
質量顕微鏡を用いた神経変性疾患細胞内封入体の解析を行うのに先立ち、これまでの研究成果を元に平成 23 年度は神経変性疾患の封入体構成タンパク質について、神経病理学的検討を進めてきた。
1) Optineurin (OPTN) E478G 変異を有する家族性 ALS の神経病理学的検討を行った。
TDP-43 陽性封入体は ubiquitin や p62 に陽性であったが OPTN には陰性であった。
TDP-43 陽性封入体を持つ脊髄前角細胞では核の TDP-43 染色性が低下していた。Golgi
装置の断片化は 70％の前角細胞に観察された。核の TDP-43 染色性が保たれているのに Golgi 装置が断片化している神経細胞も多く見られたことから、核の TDP-43 染色-73-

性低下に先行して Golgi 装置の障害が生じる可能性が示唆された。
2) FUS 変異を有する ALS や BIBD の好塩基性封入体(basophilic inclusion, BI)において、
OPTN, FUS および myosin IV が共存することを見いだした。一方 TDP-43 や SOD1 はBI と共存していなかった。これまでの結果とあわせて OPTN は TDP-43, SOD1 および
FUS という 3 つの ALS の関連タンパク質すべてと相互作用することが明らかになった。FUS は NF-B の co-activator であり、OPTN は NF-B の活性化を抑制することが知られている。FUS や OPTN が BI に補足されることにより NF-B の制御障害を来し、神経細胞変性に至る可能性がある。また OPTN と myosin IV は Golgi 装置の安定化に関わっており、これらの障害により Golgi 装置の断片化が生じて神経細胞死を来す可能性がある。
3) 進行性核上性麻痺(progressive supranuclear palsy, PSP)のリン酸化 tau 陽性封入体にSmad ubiquitination regulatory factor-2 (Smurf2)が共存していることを初めて見いだした。Smurf2 は Smad タンパクの ubiquitination を行う E3 ligase であることから、PSPの病態に TGFbeta/Smad シグナル伝達系の異常が関与していることが示唆された。
Protein disulfide isomerase (PDI)が家族性および孤発性 ALS の封入体に共存しており、PDIの機能異常が ALS でのタンパク質ミスフォールディングに関与している可能性が示唆された。

2. List of Publications
Original Articles
1. Ito H, Nakamura M, Komure O, Ayaki T, Wate R, Maruyama H, Nakamura Y, Fujita K,
Kaneko S, Okamoto Y, Ihara M, Konishi T, Ogasawara K, Hirano A, Kusaka H, Kaji R,
Takahashi R, Kawakami H. Clinicopathologic study on an ALS family with a heterozygous
E478G optineurin mutation. Acta Neuropathol. 2011 Aug;122(2):223-9.
2. Honjo Y, Kaneko S, Ito H, Horibe T, Nagashima M, Nakamura M, Fujita K, Takahashi R,
Kusaka H, Kawakami K. Protein disulfide isomerase-immunopositive inclusions in patientswith amyotrophic lateral sclerosis. Amyotroph Lateral Scler. 2011 Nov;12 (6):444-50.
Proceedings
1. Nakamura M, Kaneko S, Ito H, Asayama S, Nishii M, Fujita K, Wate R, Kusaka H
Smad ubiquitination regulatory factor-2 accumulates in TAR DNA binding protein-43 positive
cytoplasmic inclusions in spinal cord but not in hippocampus of sporadic amyotrophic lateralsclerosis 87th Annual Meeting of the American Association of Neuropathologists, Seattle
WA USA 2011/6/25-74-

Annual Report 研究成果報告書
Title of Project

Elucidation of pathogenesis of psychiatric disorder
精神障害の病態研究

P.I.

Toshihiko Kinoshita, 木下利彦

E-mailkinoshit@takii.kmu.ac.jp

Affiliation

Neuropsychiatry, 精神神経科学講座

Collaborator

加藤正樹、齊藤幸子、嶽北佳輝、坂井志帆、高野悟史

Keywords

bipolar disorder, obsessive-compulsive disorder, schizophrenia

1. SUMMARY OF RESEARCH RESULTS
Recently, biological background was found to contribute to the most of psychiatric disorder.
Among others, in schizophrenia, bipolar disorder and obsessive-compulsive disorder as well as thedevelopmental diseases such as autism and the attention deficit/hyperactivity disorder, biologicalfactor was reported to have large contribution. (Burmeister, 2008). Morbidity and heritability are
1% and 70-85% in schizophrenia, 1% and 60-85% in bipolar disorder, 1.6 % and 60-70% in
obsessive-compulsive disorder, respectively. Therefore, we wanted to touch getting a head start ofthe pathological elucidation in psychiatric disorder by using molecular imaging technique. At thepresent, the progress of this study for the each disease is as follows.
1. Schizophrenia We perform two following studies for the pathological elucidation of patientswith schizophrenia.a. Mirror neuron abnormalities in patients with first-episode schizophrenia We
succeed a three-dimensional construction of the mirror neuron using diffusion tensor image
analysis in 2011. The purpose in the future is to identify and to evaluate mirror neuron integrity inpatients with schizophrenia compared with healthy controls.b. Open label randomized trial oftreatment by second generation antipsychotics for schizophrenia patients considering the drug
susceptibility gene We completed the recruiting of the patients that it was thought that it wasnecessary in this study in 2011. We will continue the analysis of obtained data in future.
2. Bipolar disorder We plan a study aiming at the pathological elucidation of patients withJapanese bipolar disorder and a construction of the treatment algorithm. Now, we prepare for themulticenter study in this field and conduct adjustments of the protocol.
3. Obsessive-compulsive disorder We plan a study aiming at the pathological elucidation ofpatients with Japanese obsessive-compulsive disorder and a construction of the treatment
algorithm.
近年、精神障害の多くには生物学的背景が存在することが明らかになっている。
Burmeister（2008）らの報告では、精神科領域において、自閉症や注意欠陥多動性障害な
どの先天的に発症が認められる発達障害を除くと、最も生物学的背景との関連が強く認められる疾患は統合失調症、双極性障害、強迫性障害の 3 疾患であることが示されている。
それぞれの罹患率と遺伝率は、統合失調症：1%/70～85%、双極性障害：1%/60~85%、強迫性障害：1.6%/60~70%とされている。
今回我々はこれらの生物学的背景が他の精神疾患と比較して強いと考えられる疾患の病態について、分子イメージング手法を利用することで病態解明の先鞭を付けたいと考えた。
現時点での各疾患に対する本研究の進行状況は以下の通りである。
1.統合失調症
統合失調症患者の病態解明に向けて以下の 2 つの研究を行っている。
①ミラーニューロンの形態変化：拡散テンソル画像解析による精神科治療効果判定②統合失調症の個別化治療アルゴリズム構築に向けた第 2 世代抗精神病薬の無作為比較
-75-

試験
①については拡散テンソル画像解析を用いて、社会的認知と関与が深いとされているミラ
ーニューロンの三次元構築を試み、平成 23 年度に成功している。今後はどのような治療がミラーニューロンに作用し、患者の社会的認知の回復につながっているかについて検討を行っていく。
②については平成 23 年度は本研究において必要と考えられた患者のリクルートを終了した。今後は得られたデータの解析を継続して行っていく。
2.双極性障害
日本人双極性障害患者の病態解明及び治療アルゴリズムの構築を目指した研究を予定している。本分野では多施設共同試験を予定しており、現在その施行に向けたプロトコールの調整等を行っている。
3.強迫性障害
日本人強迫性障害患者の病態解明及び治療アルゴリズムの構築を目指した研究を予定している。

2. List of Publications
Books
1. 加藤正樹、木下利彦 (樋口輝彦、野村総一郎、加藤忠史); 第 11 章 うつ病の薬物療法.
うつ病の事典 うつ病と双極性障害がわかる本; 106-114,日本評論社 2011 164 頁
2. 加藤正樹; [Ⅰ.統合失調症] 4.薬物遺伝学からみた合理的な薬物選択は？. EBM 精神疾患の治療 2011-2012;28-33,中外医学社 2011 388 頁
Proceedings
1. 加藤正樹; 治療抵抗性うつ状態 predict, prevent and treat.第 30 回躁うつ病の薬理・生化学的研究懇話会 2011.7(京都)2. 齊藤幸子・Mirror neuron abnormalities in Patients with First-Episode Schizophrenia 広島精療精神医学研究会・広島・2011.10
3. 齊藤幸子、大塚達以、木下利彦、Shenton Martha・ミラーニューロンと共感～拡散テン
ソル画像解析によるミラーニューロンの三次元構築の試み～・第 13 回日本サイコセラピー学会・大阪・2012.3
4. 齊藤幸子、大塚達以、木下利彦、Shenton Martha・拡散テンソル画像解析によるミラーニューロンの三次元構築の試み・第 128 回 関西医科大学学内学術集談会・大阪・2012.3
5. 嶽北佳輝, 加藤正樹, 坂井志帆, 諏訪梓, 分野正貴, 奥川学, 木下利彦;日本人統合失調症患者に対する perospirone と aripiprazole のオープンラベル無作為割付比較試験.第 21回日本臨床精神神経薬理学会,第 41 回日本神経精神薬理学会合同年会 2011.10(東京)
6. Takekita Y, Kato M, Wakeno M, Sakai S, Suwa A, Nishida K, Okugawa G, Kinoshita T; A
Open-label, Randomized Comparative Study of Perospirone and Aripiprazole in Japanese
Schizophrenia Patients.2nd Congress of AsCNP 2011.9(Seoul)
7. Takekita Y, Kato M, Sakai S, Suwa A, Okugawa G, Kinoshita T; Comparison of Perospirone
and Aripiprazole: A 12 weeks, Randomized Open-label Study in Japanese Schizophrenia
Patients. 24th ECNP Congress 2011.9(Paris)-76-

Annual Report 研究成果報告書

Title of ProjectPossible role of propofol’s cyclooxygenase-inhibiting property in alleviating
dopaminergic neuronal loss in the substantia nigra in an MPTP-induced murine
model of Parkinson’s disease
プロポフォールの活性化ミクログリア依存性神経細胞傷害に及ぼす影響

P.I.

Koh Shingu, 新宮興

Affiliation

Medical Control of Stress Response, 侵襲反応制御学(麻酔科学)講座

Collaborators

稲田武文、久保古寿江

Keywords

intravenous anesthesia, microglia, neurodegenearative disorders, Parkinson’
disease, propofol

E-mailshingu@hirakata.kmu.ac.jp

1. SUMMARY OF RESEARCH RESULTS
Propofol is an intravenous anesthetic widely used for sedation and general anesthesia. We
investigated the effect of propofol on prostanoid production by activated microglias.
Primary microglial culture was obtained from the brains of neonatal C57BL/6 mice. The
microglias were stimulated with lipopolysaccharide (LPS) in the presence of propofol. Propofol
suppressed the LPS-induced production of prostaglandin E2 and thromboxane B2. Cyclooxygenase(COX) protein expression and arachidonic acid release were not affected by propofol, while COX
enzyme activity was significantly inhibited by propofol. The COX-inhibiting activity was also
observed with purified enzymes, with COX-2 inhibition being significantly greater than COX-1
inhibition.
Next, we studied whether the COX-inhibiting activity of propofol resulted in dopaminergic
neuroprotection in a 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) murine model ofParkinson’s disease, in which COX inhibitors, such as non-steroidal anti-inflammatory drugs, arereported to be neuroprotective. C57BL/6 mice received intraperitoneal injections of MPTP with orwithout propofol treatment, and the dopaminergic neurons in the substantia nigra pars compacta(SNpc) were examined immunohistochemically by observing the tyrosine hydroxylase-positive
cells. The number of dopaminergic neurons in the SNpc was significantly reduced by MPTP
treatment, while the MPTP-induced neuronal loss was minimal upon treatment with propofol or
the selective COX-2 inhibitor, NS-398. These results indicate that propofol might be beneficial in
mitigating MPTP-induced dopaminergic neurons, possibly via its COX-inhibiting activity.
In this era of longer life spans, the number of patients afflicted by neurodegenerative diseases
such as Parkinson’s disease and Alzheimer's disease has been increasing, as has the number ofsurgeries performed on such patients. There are two kinds of general anesthesia technique: oneuses inhalational anesthetic and the other intravenous anesthetic. Propofol is one of theintravenous anesthetics. Until now, the choice of anesthetic is largely dependent on theanesthesiologist’s preference, and not necessarily on the patient’s neurodegenerative disorders.
Our findings might be taken into account when considering what anesthetic is indicated during asurgical procedure.
プロポフォールは現在世界で最も広く使用されている麻酔薬の一つである。また近年高齢化の影響もあり、アルツハイマー病やパーキンソン病などといった中枢神経変性疾患を-77-

患った人々が増加しておりそれに伴い同名疾患を抱えた患者の手術麻酔件数も増加してきている。われわれは中枢神経変性疾患での炎症メディエーター産生細胞としてのミクロ
グリアに注目し、プロポフォールが活性化ミクログリアからのプロスタグランディンE2(PGE2) の産生を抑制することを見出した。
つぎに PGE2 合成の重要な酵素であるシクロオキシゲナーゼ (COX) に注目し、プロポ
フォールがCOX の活性を直接抑制することを見出した。したがって、プロポフォールには非ステロイド系抗炎症薬 (NSAID) 様の効果もあるようである。
また NSAID がアルツハイマー病やパーキンソン病などの中枢神経変性疾患の予防効果があることからプロポフォールとパーキンソン病との関係を調べた。具体的には MPTP
誘発性パーキンソン病モデルマウスを用いて、プロポフォールの COX 活性抑制作用が黒質ドパミン神経保護作用を示すかどうかを調べた。プロポフォールならびに COX-2 選択的阻害薬は、神経傷害を有意に抑制した。さらにプロポフォールは COX-2 選択的阻害薬と同様に黒質の MPTP 誘発性のミクログリアの活性化を抑制した。その結果プロポフォ
ールは COX 活性抑制を介し、MPTP 誘発性黒質ドパミン神経傷害を抑制する可能性があることがわかった。
麻酔薬にドパミン神経保護作用があることは、これまで知られておらず、中枢神経変性疾患の麻酔薬の選択の際の一つの指標となりうるものと考えられる。

2. List of Publications
Original Articles
1. Kubo K, Inada T, Shingu K. Possible role of propofol’s cyclooxygenase-inhibiting property inalleviating dopaminergic neuronal loss in the substantia nigra in an MPTP-induced murine
model of Parkinson’s disease. Brain Res. 1387, 2011, 125-133.
Proceedings
1. 久保古寿江、稲田武文、新宮興

パーキンソン病患者の麻酔にプロポフォールは有用か？日本臨床麻酔学会第 31 回大会

沖縄コンベンションセンター-78-

2011 年 11 月 3 日

Annual Report 研究成果報告書
Title of Project

Analysis of the Brain Digitalislike Substance Underlying HYPERTENSION
高血圧の病態解明のための脳内内因性ジギタリスの局在分析

P.I.

Hakuo Takahashi, 高橋伯夫

E-mailtakahash@hirakata.kmu.ac.jp

Affiliation

Clinical Sciences and Laboratory Medicine, 臨床検査医学講座

Collaborators

小宮山豊、吉賀正亨、横井豊彦

Keywords

central nervous system, endogenous digitalis, hypertension, hypothalamus,
sympathetic nerve activity

1. SUMMARY OF RESEARCH RESULTS
We have been searching for the endogenous digitalis as a cause of hypertension related toexcess intake of sodium for many years. It is well known that the endogenous digitalis is produced
upon sodium loading not only in rats but also in humans. When we used anti-ouabain or –digoxin
antibody to stain the immunoreactive substances in the brain, those were clearly demonstrated in
the paraventricular and supraoptic nuclei in the hypothalamus. Those nerve fibers extended into
posterior pituitary. Therefore, endogenous digitalis may be produced in the hypothalamus, andwould be released by sensing accumulation of sodium in the body. This is our working hypothesis
based on the earlier findings obtained mainly by us. To prove this hypothesis, we are aiming tofigure up the whole story of mechanisms of hypertension in special reference to endogenous
digitalis.
Recently, we found that bufadienolides are existed in the adrenal medulla and cortex, and therelease was regulated by the renin-angiotensin axis. Since the adrenal medulla is of neural crest
origin, marinobufagenin and/or marinobufotoxin may also exist in the central nervous system.
Although we explored the existence of marinobufagenin-like immunoreactivity in the
hypothalamus, it wasn’t clear to visualize so far. Metabolism of ouabain in the brain is alsosuggested to be regulated via renin-angiotensin-aldosterone system. A cell line, N1 cells,
originated from the paraventricular nucleus in the hypothalamus is now available. By using thisimmortalized cell line, we explored the possible mechanism of the production and release ofouabain; addition of aldosterone in the culture medium, dose-dependently increased the release ofouabain from N1 cells under the serum-free culture environment. This response was blunted byaddition of eplerenone, a mineralocorticoid receptor blocker, in the culture medium. Therefore,
aldosterone will be the direct agonist for secretion of ouabain.
Ouabain, digoxin and marinobufagenin, all are small molecules suitable for mass-spectrometricanalysis. We have been using this method to identify these steroids. Rapid progress in imaging
mass spectrometry may assist us to clarify the definite intracellular localization of endogenous
digitalis in the brain. We are now aiming at the project by using the novel device.
平成 18 年以前から、この研究テーマに取り組んでいて、数多くの成果を挙げている。
その結論は、交感神経調節の上位中枢である視床下部で、食塩負荷により産生されるウア
バインやマリノブファゲニンなどの内因性ジギタリス様物質が交感神経活動を亢進させることで高血圧がもたらされるとするものである。ただし、この事実については、ヒトでは証明されていないだけでなく、実験動物においてもその詳細な点では、未だ明確とは言い難い。例えば、ウアバイン、ジゴキシン、マリノブファゲニン以外のブファジェノライ
ドのいずれが主体的に役割を演じているのかは定かではない。ここ数年の間に、我々の研究グループは、平成 18 年～19 年に、世界で初めてマリノブファゲニンにスベロイルアル
ギニンが付いたマリノブフォトキシン（MBT）を脊椎動物（ラット）で発見し、これに-79-

昇圧作用があることを明らかにした。また、下垂体由来の細胞株 GH3 培養系にて MBTの分泌動態を調べたが明らかな変化はなかった。しかし、副腎皮質細胞由来の Y-1 細胞培養系では、MBT を産生していることを明らかにした。また、特筆すべきは、副腎髄質由来細胞株である PC12 細胞培養系でも MBT を産生していることを質量分析系を用いて明らかにしている。平成 20 年には、Y-1 細胞培養系で、MBT の産生と放出にアンジオテン
シン II が関与することを AT-1 受容体拮抗薬（ARB）を用いて明らかにした。そこで、自然発症高血圧ラット（SHR）を用いて、食塩負荷し、MBT の産生に及ぼすレニン－アン
ジオテンシン（RA）系の関与を検討した。その結果、食塩負荷で MBT は血圧とともに著明に増加し、ARB は昇圧を明らかに抑制するが、MBT の産生に関してはやや減少傾向はあるものの食塩負荷による増加を完全には抑制しなかった。したがって、MBT の vivo での産生における RA 系の関与は不明である。

2. List of Publications
Original Articles
1. Takahashi H, Yoshika M, Komiyama Y, Nishimura M. The central mechanism underlying
hypertension: a review of the roles of sodium ions, epithelial sodium channels, therenin-angiotensin-aldosterone system, oxidative stress and endogenous digitalis in the brain.
Hypertension Res. 34(11);2011:1147-1160.
2. Yoshika M, Komiyama Y, Takahashi H. An ouabain-like factor is secreted from immortalized
hypothalamic cells in an aldosterone-dependent manner. Neurochemistry international
59(2);2011:104-108.
3. Yoshika M, Komiyama Y, Takahashi H. Isolation of marinobufotoxin from the supernatant of
cultured PC12 cells. Clinical and experimental pharmacology & physiology.
38(5);2011:334-337.
Proceedings
1. 吉賀正亨, 小宮山豊, 高橋伯夫: 高血圧発症機序における内因性ジギタリスの臨床検
査学的研究. 第 58 回日本臨床検査医学会学術集会. 岡山、2011 年 11 月
2. 高橋伯夫: 内因性ジギタリスを中心とした血圧の中枢性調節と高血圧発症機序. 第 15回日本心血管内分泌代謝学会総会. 大阪、2011 年 11 月

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Annual Report 研究成果報告書
Studies on cell treatment for complete spinal cord injury
脊髄損傷に対する細胞治療の実験的研究

Title of Project
P.I.

Toshio Nakatani, 中谷壽男

E-mailnakatant@takii.kmu.ac.jp

Affiliation

Spinal cord Regeneration, 脊髄再生医学（救急医学科）

Collaborators

岩瀬正顕、前田裕仁、齊藤福樹、津田雅庸

Keywords

bone marrow stromal cells, cell infusion therapy, spinal cord regeneration,
spinal cord injury

1. SUMMARY OF RESEARCH RESULTS
We have been studied experimental and clinical research for the treatment of spinal cord injury.
This year, we have made our effort to summarize and publish our recent clinical work as follows:
To determine whether intrathecal administration of cultured autologous bone marrow stromal cells(BMSCs) is safe and feasible for treatment of subacute spinal injury. Five patients with complete
tetraplegia due to cervical spinal injury on admission were included. A small amount of bonemarrow was obtained during surgery for spinal fusion. BMSCs were cultured, reaching 107-108
cells. The properties and functional efficacy of the BMSCs were verified with surface marker
analysis and a neurite extension test. BMSCs were administered by lumbar puncture. The patientswere closely observed for 6 months, and the Committee on Effectiveness and Safety of ClinicalTreatment (CESCT) evaluated safety. No adverse responses were observed in biochemical andradiographic examinations. The CESCT did not recognize any harmful effects of thetransplantation, and concluded it was safe for treatment. The patients were further followed up for
1 to 4 years with no adverse responses. The recovery of American Spinal Injury Association
Impairment Scale (AIS) B and C patients at transplantation was rapid and remarkable, but gradualor limited in AIS A patients.

This study demonstrated that intrathecal administration of cultured

autologous BMSCs is safe and feasible for treatment of spinal cord injury.

The above

study report has been accepted for publication. In our next step, we are planning to administerbone marrow mononuclear cells of the patient without incubation and proliferation. We alsoplanning an experimental study to continue our clinical trials.
我々は、従来より脊髄損傷の治療に向けての実験的研究、臨床試験を行ってきた。今年
度はそれらのうち、臨床試験の成果をまとめ、公表することに精力を注いだ。
我々がこれまで行ってきた培養自己骨髄間質細胞の髄液内投与による脊髄損傷治療の安全性と実現性について検討するために、頸髄損傷による四肢完全麻痺の５症例に実施した臨床試験成績をまとめて見た。試験に於いては受傷後の頸椎固定手術に際して、少量の骨髄海綿骨を採取し、培養して間質細胞を 107 乃至 108 に増殖させ、その細胞の質に関しては表面マーカーと突起伸長試験を経て確認した後に、患者の髄液内に投与して、移植細胞を損傷脊髄に到達せしめ 6 ヶ月の試験期間、及びそれ以降も経過を追跡し、外部委員を含む効果安全性評価委員会が安全性について評価した。血液生化学検査にて、或いは画像診断にて追跡しているが、これまでのところ最長 4 年間を経過しているが、何ら骨髄細胞移植によると思われる有害反応は無く、機能的には一症例で歩行可能、一症例で装具にて起立保持可能、2 例にて車椅子の自己操縦可能となり、American Spinal Injury Association-81-

Impairment Scale にても motor score は従来の治療を行った症例に比べて、著しい改善を認めた。ただし、1 例のみは無効であった。本臨床試験は安全であり、かつ実現性が可能なものと考えている。
現在、我々は本治療法を確立するためにはより多施設での臨床試験が必要と考え、そのためには骨髄細胞を培養することなく、分離後直ちに移植する方法での臨床試験を行うべく、動物実験を経て臨床試験の準備を行っている。

2. List of Publications
Original Articles
1. Saito, F., Nakatani, T., Iwase, M., Maeda, Y., Murao, Y., Suzuki, Y., Fukushima, M., Ide, C.
Administration of cultured autologous bone marrow stromal cells into cerebtospinal fluid inspinal injury patients: a pilot study. Restor. Neurol. Neurosci. 30: 127-136, 2012.

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Division of Cancer

がん部門

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Annual Report 研究成果報告書
Title of Project

Dynamics of adult tissue specific stem cells by multi-color lineage tracing
method
多色細胞系譜追跡法を用いた幹細胞の維持機構の解析

P.I.

Hiroo Ueno, 上野博夫

E-mailhueno@takii.kmu.ac.jp

Affiliation

Stem Cell Pathology, 幹細胞病理学講座

Collaborators

比舎弘子、神田晃、槇政彦、田中敏宏、駒井資弘、矢内洋次、大町太一、
大江秀一

Keywords

development, regeneration, stem cell

1. SUMMARY OF RESEARCH RESULTS
We have developed a novel method for lineage tracing by using the cre-loxp-mediatedmulticolor mosaic system (2010 Nature, Red-Horse and Hiroo Ueno, 2011 Nature, Yuval
Rinkevich and Hiroo Ueno). On the other hand, it has been reported that there are two kinds ofstem cells in intestinal crypt, long-term slow growing stem cells, and short-term rapidly
proliferating stem cells. We have already got cre-knockin mice that can detect these stem cell
populations. We have also succeeded in establishing the in vitro culture method of intestinal
epithelial cells. By combining these techniques, we are currently analyzing dynamics of two kindsof intestinal stem cells by time lapse imaging on various conditions.
事業推進者はマルチカラー細胞系譜追跡法を開発して消化管上皮細胞の発生、成体腸幹細胞の維持機構について解析を行って来た。現在、大腸がん発症モデルを用い、そのがん化過程における正常成体腸幹細胞の維持機構の破綻について imaging を行っている。
大腸がんの前がん病変である大腸腺腫の発症にはがん抑制遺伝子である adenomatous
polyposis coli (Apc) 遺伝子の変異が深く関与している事が知られている。ヒト大腸がんにおいて Apc 遺伝子の変異または欠失が高頻度に認められ、また家族性ポリポーシスの家系に Apc 遺伝子の生殖細胞系列変異が報告されており、マウスにおいても Apc に点突
然変異を持つ Apcmin マウスなどが家族性ポリポーシスモデルマウスとしてよく知られている。Apc 遺伝子産物は腸幹細胞の増殖シグナル伝達経路の一つである Wnt シグナル伝達経路の抑制因子であり、その変異により Wnt シグナルが異常に活性化される事が腸幹細胞の異常増殖につながり、腺腫の発症に関与すると考えられている。現在これらのモデルマウスを使いて、幹細胞を多色標識し、その動態について詳細にイメージングを行っている。
イメージング技術の向上により研究効率が上昇した。３D ソフトウェアにより画像の立体像が解析出来る様になり、幹細胞の動態が正確に把握できるようになった。オールインワン顕微鏡の導入により Time lapse imaging が可能となり、単一幹細胞由来の細胞集団
を時間経過とともに追える様になった。

2. List of Publications
Original Articles
1. Rinkevich Y., Lindau P., Ueno H., Longaker, M., Weissman IL., Germ layer and lineage
restricted stem/progenitors regenerate the mouse digit tip. Nature, 476, 409-413.
Proceedings
1. 上野博夫・多色細胞系譜追跡法による成体幹細胞解析法・日本病理学会・横浜・2011
2. 上野博夫・マルチカラー細胞系譜追跡法を用いた幹細胞・発生研究の現状・日本サイ
トメトリー学会・京都・2011-84-

Annual Report 研究成果報告書
Title of Project

Analysis of ATL development in HTLV-1 infected humanized mouse model
HTLV-1 感染ヒト化マウスを用いた ATL 発症過程の解析

P.I.

Jun-ichi Fujisawa, 藤澤順一

E-mailfujisawa@takii.kmu.ac.jp

Affiliation

Viral Oncology, ウイルス腫瘍学（微生物学）講座

Collaborators

竹之内徳博、田中正和、上野孝治、手塚健太、恩額日楽、荀潤澤Keywords

ATL, HTLV-1, humanized mice, leukemia stem cell

1. SUMMARY OF RESEARCH RESULTS
Humanized mice (huNOG) established by the intra-bone marrow transplantation of NOG-SCID
mouse with CD133+ hematopoietic stem cells purified from human cord blood were infected with
HTLV-1 in vivo by peritonial injection of -ray-irradiated MT-2 cells in 3 to 4 months after
transplantation.
While normal differentiation of human T lymphocytes was observed in the spleen of uninfected
huNOG mouse, HTLV-1 infection increased the number of CD25+ CD4+ T- lymphocytes andresulted in the splenomegaly within several months. In the late period of infection, where almost
all of the blood cells in the mouse were composed of infected human T-lymphocytes, cells withhighly lobulated or flower-shaped nuclei appeared in the peripheral blood.
Inverse PCR analysis of provirus integration sites revealed the polyclonal infection in the earlyphase of infection and the oligoclonal expansion of infected T cells mostly in the population of
CD25+ CD4+ T- cells in the late phase. Since substantial amount of anti-Gag antibodies andTax-specific CTLs were detected in the serum and the spleen of infected mice, respectively, theinvolvement of immune system against HTLV-1 was suggested in the clonal selection of HTLV-1
infected T-cells in this system.
When CD4+ T- cells in a spleen of infected mouse were divided into CD25+ and CD25populations, common infected cell clones in terms of provirus integration site were identified in
two populations and the percentage of the most abundant clone was larger in CD25+ cells than in
CD25- cells, indicating that the conversion from CD25- to CD25+ in infected CD4+ T-cells is
associated with the selective growth in the late phase of infection. Furthermore, expression of Tax,an oncogenic protein of HTLV-1, was observed in CD25- population but not in CD25+ population.
These results suggest that some epigenetic change in the infected CD25- cells results in theexpression of CD25 and the suppression of Tax, leading to the selective growth of infected T-cell.
Thus, the HTLV-1 infected huNOG mouse model should provide a valuable system for the
analysis of ATL pathogenesis and the development of treatments against various HTLV-1
associated diseases. Effect of in vivo administration of various anti-tumor or anti-viral drugs on
the overgrowth of infected T-cells should be assessed in future.
ヒト臍帯血由来造血幹細胞の NOG-SCID マウスへの骨髄内骨髄移植により作製したヒト化マウスの腹腔内に HTLV-1 産生細胞株を移入することにより、マウス個体内ヒト T 細胞への HTLV-1 感染と感染 T 細胞の異常増殖および脾腫・肝臓への転移等、ATL 様病態の誘導に成功した。感染数ヶ月の末梢血において、ATL 細胞特異的な花弁様分様核を持ったリンパ球の出現も再現された。
感染 T 細胞表面抗原の解析から、感染初期では CD25(-)CD4(+)T 細胞の異常増殖が観察されたが、感染後期においては ATL 腫瘍細胞と同様の CD25(+)CD4(+)T 細胞が感染細胞の大部分を占めるようになった。さらに HTLV-1 プロウイルス組み込み部位の解析から、感染の経過とともに、特定の感染クローンの選択的な増殖が観察され、白血病細胞の選択過程が再現されていると考えられた。
感染個体内における腫瘍細胞の選択的増殖の主要な原因として考えられる抗 HTLV-1 宿-85-

主免疫を解析したところ、感染ヒト化マウス血清中には抗 HTLV-1 Gag 抗体が、また、脾
臓内には感染細胞の排除に関与していることが既に示されている抗 Tax CTL の存在が確認された。
次に感染細胞の増殖と選択過程における HTLV-1 遺伝子発現の関与を解析したところ、
CD25(-)CD4(+)T 細胞では HTLV-1 の発がん遺伝子 tax の有意な発現が観察されたが、CD25(+)CD4(+)T 細胞においては tax の発現は抑制されていた。Tax 蛋白の細胞トランスフ
ォーム能と細胞増殖促進能は既に証明されていることから、感染初期では Tax の機能を介した CD25(-)CD4(+)T 細胞の異常増殖が考えられたが、感染後期においては tax 遺伝子の発現抑制と同期した CD25 の発現活性化を含む何らかの遺伝子発現の変化が CD25(+)
CD4(+)T 細胞の選択的増殖に関与していることが強く示唆された。さらに、同一感染個体
中の CD25(-)CD4(+)T 細胞群と CD25(+)CD4(+)T 細胞群における、感染クローンの解析を行ったところ、両群に共通の感染クローンの存在が示され、かつ最大クローンは CD25(+)
CD4(+)T 細胞群により高い占有率を示したことから、単一の感染 CD4(+)T 細胞クローン
が CD25(-)から CD25(+)に転換したことが明らかとなり、腫瘍細胞の選択過程における遺伝子のエピジェネティック変異の可能性が強く示唆された。

2. List of Publications
Original Articles
1. Tanaka M, Nitta T, Sun B, Fujisawa J, Miwa M. The route of primary HTLV-1 infection
regulates HTLV-1 distribution in reservoir organs of infected mice. Exp. Thr. Med., 2, 2011,89-94.
Proceedings
1. Takaharu Ueno, Kenta Tezuka, Runze Xun, Mami Tei, Masakazu Tanaka, Norihiro
Takenouchi, Jun-ichi Fujisawa：Infection of defective virus correlated with the induction of
CD25 positive CD4 T-cell during early phase of infection in humanized mouse model.
The XV International Congress of Virology, 札幌, 9 月 12 日, 2011 年
2. Kenta Tezuka, Runze Xun, Mami Tei, Takaharu Ueno, Masakazu Tanaka, Norihiro
Takenouchi, Jun-ichi Fujisawa: Inverse correlation between tax and CD25 expressions in
HTLV-1 infected CD4+ T-cells in vivo.
The XV International Congress of Virology, 札幌, 9 月 13 日, 2011 年
3. Masakazu Tanaka, Naoki Wada, Iwao Hashimoto, Sho Hasegawa, Hiroyuki Tsuda, Masanao
Miwa, Jun-ichi Fujisawa: Anti-tumor effects of bovine lactoferrin to lymphoma cells
expressing HTLV-1 Tax.
第 70 回日本癌学会学術総会、名古屋、10 月 5 日、2011 年
4. 手塚健太、上野孝治、荀潤澤、竹之内徳博、田中正和、藤澤順一：HTLV-1 感染ヒト
化マウスにおける欠損型プロウイルスの解析
第70回日本癌学会学術総会、名古屋、10月5日、2011年-86-Annual Report 研究成果報告書
Title of Project

Identification of embryonic stem cell-like tissue committed stem cells andelucidation of their differentiation pathway: An application to cell therapy andregenerative medicine
ES 細胞様組織幹細胞の同定と分化経路の解明：細胞移植・再生医療への応用

P.I.

Yoshiaki Sonoda, 薗田精昭

Affiliation

Stem Cell Biology, 幹細胞生物学(衛生学)講座

Collaborators

佐々木豊、中塚隆介、岩城隆二、高橋昌也

Keywords

ES cells, regenerative medicine, tissue stem cells, VSEL

E-mailsonoda@takii.kmu.ac.jp

1. SUMMARY OF RESEARCH RESULTS
The aim of this study is to identify embryonic stem cell-like tissue committed stem cells fromvarious murine and human tissues and to apply them for the cell therapy and regenerative medicine
in the near future. This year, we identified novel mouse dental pulp stem cell (DPSC) and analyze
their functional properties (1). Next, we developed a new method for effective isolation of mousevery small embryonic-like stem cell (VSEL) (2).
1) Identification of mouse dental pulp stem cells (DPSCs)
Using an enzymatic treatment, we succeeded to identify novel mouse dental pulp stem cells(DPSCs), which expressed PDGFR and Sca-1. These DPSCs exhibited a high growth potential
and an ability to differentiate into mesenchymal cell lineages. Proliferation and differentiation
potentials of DPSCs were improved by hypoxic culture condition. Moreover, these DPSCs could
support human primitive hematopoietic stem cells.
2) Development of a new isolation method for mouse very small embryonic-like stem cells(VSELs)We tried to isolate VSELs from murine bone marrow cells according to the reported method. We
could isolate substantial number of VSELs, which were under 10 m in a diameter. However, theisolation efficiency was very low. Then we developed a new isolation method using murine bone
tissues and enzymatic preparations. Using our new isolation method, we could constantly collect
much larger numbers of VSELs for subsequent experiments.
These isolated VSELs expressed ES cell markers, including Oct4, Nanog, Rex1, and Dppa3. The
in vivo dynamics of VSELs in liver injury and the change of VSELs in aged mice are interesting.
Now we are proceeding the functional analysis of these VSELs.
本研究では、マウス及びヒト由来の各種組織より、ES 細胞様組織幹細胞を同定し、近未来の細胞移植・再生医療への応用を目指している。今年度は、まず、マウスを用いて、
1）歯髄由来の未分化組織幹細胞の同定と機能解析、2）very small embryonic-like stem cell
(VSEL)として報告されている未分化な組織幹細胞の同定と効率的な分離方法の開発に着手した。
1）に関する成果
マウス切歯歯髄から酵素処理法を用いて細胞を分取した。これらの細胞より、
Sca-1+PDGFR+歯髄幹細胞（dental pulp stem cell, DPSC）を予期的に分離することに成功した。DPSC は、高い増殖能力を持ち、骨、軟骨、脂肪細胞への分化能を有していた。さらに、低酸素培養が DPSC の増殖・維持に有用であること、骨髄由来の間葉系幹細胞（MSC）と同様に、未分化な造血幹細胞を維持することも明らかにしている。今後、DPSC の未分化性に関して、さらに解析を進める予定である。
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2）に関する成果
既報の方法により、マウス骨髄より VSEL の分離を試みた。報告されているように、直径 10μm 以下の非常に小型で核細胞比の大きな VSEL が分離可能であったが、その採取効率は低く、定量的な解析は困難であった。そこで、種々の方法を試みる過程で、マウス骨
と酵素処理法などを工夫することにより、既報に比べて格段に分離効率のよい新たな方法の開発に成功した。
新たな方法で採取した組織幹細胞は、直径 10μm 以下の非常に小型で核細胞比の大きな細胞であり、Oct4, Nanog, Rex1, Dppa3 などを発現していた。現在、本細胞の機能解析を進めているが、肝障害時の in vivo 動態、加齢による変化など、興味ある結果を得ている。

2. List of Publications
Proceedings
1. 中塚隆介、松岡由和、岩城隆二、高橋雅也、佐々木豊、薗田精昭：歯形成端に存在する新規マウス歯髄幹細胞の予期的分離と特性の解析。第 21 回日本サイトメトリー学会
総会、京都、2011 年 6 月 26 日
2. 中塚隆介、松岡由和、岩城隆二、高橋雅也、佐々木豊、薗田精昭：マウス切歯由来歯髄幹細胞によるヒト造血幹細胞の in vitro 支持能の検討。第 73 回日本血液学会総会、名古屋、2011 年 10 月 14 日
3. 中塚隆介、松岡由和、岩城隆二、高橋雅也、佐々木豊、薗田精昭：マウス組織に由来
する間葉系幹細胞と歯髄幹細胞機能比較～ヒト造血幹細胞の in vitro 支持能に関して、第 34 回日本造血細胞移植学会総会、大阪、2012 年 2 月 25 日

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Annual Report 研究成果報告書
Title of Project

Functional analysis of overexpressing genes in tissue stem cells and relationshipwith cancer stem cells
組織幹細胞に高発現する遺伝子の機能解析とがん幹細胞との共通性
の検討

P.I.

Seiji Kanda, 神田靖士

Affiliation

Public Health, 公衆衛生学講座

E-mailkandas@takii.kmu.ac.jp

Collaborators
Keywords

auditory pathway, cancer stem cell, pituitary, side population cells, tissue stemcell

1. SUMMARY OF RESEARCH RESULTS
We focused on the tissue stem cells in pituitary and auditory pathway. In our previous study, we
identified the tissue stem/progenitor cells in pituitary gland and cochlear nuclei of auditory
pathway in vivo. In this study, we tried to characterized these tissue stem/progenitor cells andanalyze the gene expression.
Pituitary: Three days age of mice were injected BrdU to the hypodermic at 2 times/day for 3
days continuously, and sacrificed after 10 weeks to identify the slow-cycling cells suggested thepossibility of stem cells. The results of immunohistochemistry showed that a few cells wereidentified as BrdU+ and Bcrp1+ double positive cells in the section of pituitary gland.
Furthermore, we assumed that it was possible to purify stem cells as side population (SP) cells.
Pituitary cells were isolated from 6 weeks age of mice and stained with Hoechst 33342. After
staining, SP cells were sorted as a negative fraction. The population of SP cells was about 1% of
total pituitary cells. Furthermore, in order to analysis the gene expression of these cells, we usedmicroarray techniques by Mouse oligo Microarray（Agilent technologies）. The results showed thatthe upregulated genes in SP cells were included some specific markers of stem cells, Bcrp1 andSca-1 etc. These cells also had the sphere forming ability. Currently we are going to study whether
these cells have the multipotential ability as stem cells.
Furthermore, we found some pituitary specific genes by microarray and this was one of theoncogenes. Some reports showed that tissue stem cells have common points with cancer stem
cells, and this is a possibility to become a cancer from normal tissue stem cells in vivo. Currently
we are going to analyze the functions of these genes in vitro.
Auditory pathway: Slow cycling cells were localized in cochlea nuclei (CN) by the same
methods of pituitary. Bromodeoxyuridine (BrdU)-injected mice were prepared and detected by
flowcytometry. The cells were detected 1% as BrdU positive cells in CN. SP cells were detected ata frequency of 4.4% and expressed stem/progenitor markers, Abcb1b, Abcg2, Sca-1, Notch1,
Notch4, Hes1 and Jag1 with microarray. CN stem cells also had a self-renewal ability in order tobe able to form spheres continuously. Furthermore, expressions of Abcb1b, Abcg2, Sca1, Oct3/4
and Sox2 as determined by RT-PCR were supported the data of microarray. CN cells were also
had the activity of sphere formation in young mice, but this activity was decreased by aging. It is
possible that tissue stem cells are localized in CN. Currently we are going to study whether thesecells have the multipotential ability as stem/progenitor cells.
下垂体幹細胞の特徴付けと癌幹細胞との関連の検討
下垂体細胞を Hoechst33342 で染色した後、FACS にて SP 細胞としてソーティングした-89-

ところ約 1％存在し、マイクロアレイ解析にてその発現遺伝子のプロファイリングを行ったところ、幹細胞に共通して発現しているマーカー遺伝子が高発現していることを見いだした。また、その中に下垂体に特異的に発現している遺伝子が数種類見つかり、これらの一つが下垂体腫瘍細胞に特異的に発現する遺伝子であることを突き止めた。その遺伝子のいくつかは oncogene であることがわかった。近年、組織幹細胞とガン幹細胞の共通点を有するという報告があり、今後はその遺伝子の機能解析を行う予定である。
聴覚伝導路における組織幹細胞の同定と特徴付け
本研究の結果から 3 週齢マウスの蝸牛神経核に約１％の slow cycling cell が存在し、マイクロアレイの結果から Sca1、Oct3/4、Sox2、Notch signal、Hes1、Jag1 など未熟な細胞に共通でみられる遺伝子の発現が MP 細胞と比較して高発現していた。中でも、Ndrp
や Ntf（neurotrophin）3 など neuron と分化に関連のある遺伝子が検出されたことから新規
組織幹細胞のマーカーとなる可能性が考えられた。さらに蝸牛神経核の SP 細胞は sphere形成能を有し、この活性は加齢とともに低下した。これらのことから聴覚伝導路における
蝸牛神経核には組織特異的な幹細胞または前駆細胞が存在する可能性が示唆された。

2. List of Publications
Original Articles
1. Ooka, H., Kanda, S., Okazaki, H., Suzuki, H., Mishima, K., Saito, I., Yagi, M., Tomoda, K.,
Nishiyama, T. Characterization of side population (SP) cells in murine cochlear nucleus. Acta
Oto-Laryngologica, 2012 (in press)
2. Osumi, Y., Shibata, S. B., Kanda, S., Yagi, M., Ooka, H., Shimano, T., Mikiya A., Kawamoto,K., Kuriyama, H., Inoue, T., Nishiyama, T., Yamashita, T., Tomoda, K. Downregulation of
N-methyl-D-aspartate receptor ζ1 subunit (GluN1) gene in inferior colliculus with aging.
Brain Research 2012 (in press).
Proceedings
1. S Kanda, H Ooka, H Okazaki, H Suzuki, T Nishiyama. Preparation and characterization of
pituitary stem progenitor cells in mouse. 9th Annual Meeting, International Society for
Stem Cell Research (Tront, Canada). 平成 23 年 6 月
2. H Ooka, S Kanda, T Nishiyama, K Tomoda, H Okazaki. Identification of tissue specific
Stem/Progenitor Cells in Auditory Pathway. 9th Annual Meeting, International Society
for Stem Cell Research (Tront, Canada). 平成 23 年 6 月-90-Annual Report 研究成果報告書
Title of Project

Molecular mechanism of lymphocyte trafficking
リンパ節のホーミングの分子機構に関する研究

P.I.

Tatsuo Kinashi, 木梨達雄

E-mailkinashi@takii.kmu.ac.jp

Affiliation

Molecular Genetics, 附属生命医学研究所

Collaborators

片貝智哉、植田祥啓、羽廣克嘉、富山尚、安田鐘樹

Keywords

antigen receptor, cell migration, chemokine, integrin

分子遺伝学部門

1. SUMMARY OF RESEARCH RESULTS
Immune cell trafficking plays important roles in host defense and immunological diseases.
Intracellular signaling of small GTPases Rap1 through RAPL and Mst1 regulates adhesive
processes during lymphocyte trafficking mediated by integrins. Lymphocyte interactions withhigh endothelial venules is mediated by the adhesion cascade. Using knockout and knockdown
approach, we demonstrate requisite involvement of Rap1 in the arrest step, which is stabilized
within several seconds by RAPL and Mst1 as well as talin. Two-photon microscopy is powerfultool to dissect lymphocyte behaviors after entry into lymphoid tissues. So far most of the study to
examine T cell migration within LN is to observe T cells transferred intravenously before
two-photon imaging. The major caveat of these study is that the same chemokines and integrins(CCL19/CCL21 and CCR7 or LFA-1 and ICAM-1) are also required for entry into LN. In
addition, T cells derived from knockout mice deficient for integrins and chemokines is potentiallyproblematic, since deficiencies for these molecules impair not only trafficking but also T cellhomeostasis, often leading to immune diseases. To circumvent these issues, we developed thelymph node tissue slice method, so that lymphocytes can be directly applied to the LN
parenchyma and evaluate acute effects of blocking of integrins and chemokine signaling with
antibodies and chemical inhibitors. Two-photon imaging demonstrates a distinct role of LFA-1 andICAM-1 of lymphocyte migration within LN. ICAM-1 is expressed on fibroblastic reticular cells(FRC) that constitute the T cell zone, and required for rapid, directional migration of T cells.
Mst1-deficient T cells or LFA-1 blockade exhibited slow migration with unstable interactions with
FRC network. Mst1-deficient T cells exhibited slow migration within LN. When encountered with
antigen-presenting cells, Mst1-deficient cells did not stop efficiently and exhibited unstableassociation. Thus, Mst1 is required for stable interactions with antigen-presenting dendritic cells,
which is consistent that LFA-1 and ICAM-1 is required in T-APC interactions.
The process of thymocyte selection and development is regulated by not only affinity of TCR but
also cell trafficking and cell-cell contact. Recently We have found Mst1 deficiency impairs
thymocyte selection as well as generation of FoxP3+ Treg in the thymus. To identify the roles for
integrins and Mst1 in thymocyte trafficking and selection process, we are currently examining
thymocyte trafficking within thymic tissues with 2-photon microscopy.
低分子量Ｇタンパク質 Rap1 と Rap1 結合分子 RAPL、およびその下流エフェクター分子として作動する Ste20-like キナーゼ Mst1 による接着分子 LFA-1 の制御と免疫系細胞の移動について 2 光子顕微鏡を用いたイメージングを行った。これまでノックアウトマウスの解析などによって Rap1 シグナルがもつ免疫細胞の動態調節の重要性として、リンパ節内のリンパ球移動や抗原提示細胞との相互作用が考えられることから、in vitro や組織スライスを用いた研究をおこなった。これらの系を用いた解析から、従来リンパ球移動における役割が不明確であった間葉系の細網細胞（fibroblastic reticular cells, FRC）がリンパ節内の移動において重要な役割を持つことが明らかになった。また、Mst1 がリンパ球が抗原提示細胞に遭遇し、抗原受容体によって異物由来抗原に結合すると、免疫シナプスを形成するが、Mst1 欠損リンパ球ではその接着が不安定であることを明らかにした。Mst1 欠損-91-マウスでは自己免疫様病態が加齢とともに顕著になるが、胸腺の選択に異常があることが明らかになった。胸腺組織スライスを用いて胸腺細胞の組織内動態を観察する手法を確立
し、細胞移動や組織特異抗原を発現する胸腺上皮細胞との相互作用を現在、2 光子顕微鏡等を活用して調べている。

2. List of Publications
Original Articles
1. Harada, Y., Tanaka, Y., Terasawa, M., Pieczyk, M., Habiro, K., Katakai,T.,
Hanawa-Suetsugu, K., Kukimoto-Niino, M., Nishizaki, T., Shirouzu, M., Duan, X., Uruno, T.,
Nishikimi, A., Sanematsu, F., Yokoyama, S., Stein, J.V., Kinashi, T., Fukui, Y. DOCK8 is a
Cdc42 activator critical for interstitial dendritic cell migration during immune responses.
Blood. In press.(2012)
2. Hnanawa-Suetsugu, k., Kukimoto-Niino, M., Mishima-Tsumagari, C., Akasaka, R., Ohsawa,N., Sekine, S., Ito, T., Tochio, N., Koshiba, S., Kigawa, T., Terada, T., Shirouzu, M.,
Nishikimi, A., Uruno, T., Katakai, T., Kinashi, T., Kohda, D., Tukui, Y., Yokoyama, S.
Structural basis for mutual relief of the Rac guanine nucleotide exchange factor DOCK2 andits partner ELMO1 from their autoinhibited forms. Proc Natl Acad Sci USA. 28:109（9）:3305-10(2012)
3. Kinashi, T. Overview of integrin signaling in the immune system. Methods Mol. Biol.
757:261-78 (2012)
Proceedings
1. 木梨達雄 Rap1 シグナルによるリンパ球動態の制御機構 新学術研究「動く細胞と場のクロストークによる秩序の生成」第 1 回 公開シンポジウム 「動く細胞と場を読む」
2012 年 1 月 名古屋
2. Kinashi Tatsuo, Regulation of immune cell trafficking and antigen recognition through Rap1
signaling The 21st Hot Spring Harbor Symposium jointly with 9th Global COE International
Symposium: Cell Migration in Biology and Medicine, Fukuoka Japan, Jan 2012.
3. Kinashi Tatsuo Regulation of thymocyte trafficking and antigen recognition within thymic
tissues through integrins and Rap1 effector Mst1. Keystone Symposia on Molecular andCellular Biology Chemokines and Leukocyte Trafficking in Homeostasis and Inflammatio,
Breckenridge USA, Jan 2012
4. 木梨達雄、片貝智哉、植田祥啓、羽廣克嘉 イメージングによるリンパ球組織内移動の調節とその破綻の解析 第 61 回日本アレルギー学会秋季学術大会 2011 年 11 月
東京
5. Katakai Tomoya,Tatuso Kinashi, High-speed interstitial T cell migration within lymph node
parenchyma involves LFA-1-dependent and -independent motility modes. 2011 The Annual
Meeting of the Japanese Society for Immunology, Chiba Japan, Nov. 2011
6. Ueda Yoshihiro, Katagiri Koko, Tomiyama Takashi, Yasuda Kaneki, Habiro Katsuyoshi,
Katakai Tomoya, Ikehara Susumu, Kinashi Tatsuo, Mst1 regulates thymocyte trafficking andantigen recognition within thymic tissues. 2011 The Annual Meeting of the Japanese Society
for Immunology, Chiba Japan, Nov. 2011
7. Habiro Katsuyoshi, Katakai Tomoya, Kinashi Tatsuo, Two-step model of T cell-DC primarycontacts and roles of integrin-regulatory Mst1.2011 The Annual Meeting of the JapaneseSociety for Immunology, Chiba Japan, Nov. 2011
8. 木梨達雄
接着制御シグナルの破綻と自己免疫疾患
第二回シンポジウム 2011 年 9 月 東京

ＣＲＥＳＴ「免疫機構領域9. Kinashi, T., Katagiri, K., Katakai T., Ueda, Y., Habiro, K. Crucial roles of Mst1 and RAPL
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(RASSF5) in lymphocyte adhesion and proliferation. 2nd International RASSF Symposium,
Oxford UK, July 2011
10. Kinashi, T., Katagiri, K., Katakai T., Ueda, Y., Habiro, K. Cell migration and antigen
recognition within lymph nodes and thymus. London Research Institute Cancer Research UK,
London UK, July 2011-93-

Annual Report 研究成果報告書
Title of Project

Generation and image analysis of a gene-modified animal model
疾患モデル動物の作製およびイメージング

P.I.

Sung-il Lee, 李成一

E-mailAffiliation

Model Animals, モデル動物部門

Collaborators

小沢まどか

Keywords

gene-engineered animals

silee@takii.kmu.ac.jp

1. SUMMARY OF RESEARCH RESULTS
In the immune system, cell adhesion molecules enhance the efficiency of specificreceptor-dependent lymphocyte-accessory cell and lymphocyte-target cell interactions; they arealso important in leukocyte-endothelial cell interactions and lymphocyte recirculation. The LFA-1
which is one of the adhesion molecules, is controlled by small GTPase Rap1, RAPL (the Rap1
counterpart molecule), and its downstream effector molecule, Mst1. Previous studies using theirgene knockout mice, suggested a critical role for LFA-1-associated signaling pathways inlymphocyte migration or their arrest. Actually, we observed that Rap1 adhesion activity to
ICAM-1 was suppressed in MST1-deficient and RAPL-deficient dendritic cells, by image analysis
of cell-adhesion molecule interactions. We built the targeting vector construction for
Rap1-deficient, and the construct was carried out to embryonic stem cell originated from C57BL/6
mouse origin, which newly established by our laboratory. These generated chimera mice werebrown to obtain offspring that are heterozygous for the Rap1 knockout allele. In present, we arejust going to perform the germ-line transmission ability of these chimera mice.
The import approach of the immune cells from a vein passes through two steps of a lymph vessel
to lymph nodes and to lymph vessel from a high endothelial venules. In this case, it is difficult for
specify the stage which influenced by change of the dynamic state of the Rap1-deficient cells. As
a future schedule, we have to analyze the phenotype of the obtained Rap1-deficient mouse using
molecular biological, biochemical and histological methods, for our knowledge ofRap1-associated immune cell dynamic events in vivo. Also, we will try the introduction of the
Rap1-labeled immune cells from a vein of mice, and are due to be observed that the motion of thelymphocyte within lymph nodes by imaging using 2 photon laser microscope.
免疫機構は、リンパ節内のリンパ球の移動や抗原提示との相互作用などの、免疫細胞の動態の調節を基盤にして作動している。接着分子のひとつである LFA-1 は、低分子量 G
たんぱく質 Rap1 と Rap1 結合分子 RAPL、その下流エフェクター分子として作動する Mst1
によって制御されており、これらのシグナル経路は今までのノックアウトマウスの解析などから、免疫細胞の動態調節に重要であることがわかっている。実際、MST1 や RAPL が欠損している樹状細胞では、ICAM-1 との接着の際に Rap1 の活性化が落ちる傾向にあることを、イメージングを用いた実験で観察している。
今後の予定として、得られた Rap1 遺伝子欠損マウスの表現型を分子生物学的・生化学
的・組織学的に解析し、in vivo での Rap1 遺伝子の働きを特定する。またリンパ球、樹状細胞などの免疫細胞をラベルして静脈から導入し、リンパ管からリンパ節への組織移動を、2 光子レーザー顕微鏡を用いたイメージングで観察する予定である。

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Annual Report 研究成果報告書

Title of Project

Identification of causative material of small-bowel obstruction from
histopathological sections by MALDI-imaging mass spectrometry
質量顕微鏡法を用いた病理標本解析による腸管内腔閉塞原因物質の同定と発症機構の解明

P.I.

A-Hon Kwon, 権雅憲

E-mailkon@hirakata.kmu.ac.jp

Affiliation

Surgery, 外科学講座

Collaborators

海堀昌樹、山田正法

Keywords

intestinal obstruction (ileus), imaging mass spectrometry

1. SUMMARY OF RESEARCH RESULTS
Bowel obstruction or intestinal obstruction is a mechanical or functional obstruction of theintestine that prevents the normal transit of digested materials. It can occur at any levels distal tothe duodenum of the small intestine and is typically considered to represent a medical emergency.
The case in this study, surgery was needed to remove the necrotic tissue and causative materials.
Laparoscopic operation was performed to excise the inflamed small intestine and the obstructive
material. For imaging MS analysis, the surgically isolated intestinal portion and the foreign body
were fixed and paraffin embedded as performed routinely at Kansai Medical University Hospital.
Even after the materials were removed, the cause and the identity of the materials were notclarified. To obtain the information of the extirpated materials, imaging mass spectrometry (IMS)
technique was employed.
Direct tissue analysis using matrix-assisted laser desorption/ionization (MALDI)-mass
spectrometry (MS) provides the means for in situ molecular analysis of a wide variety of
biomolecules. This technology, IMS, allows measurement of biomolecules in their native
biological environments without the need for target-specific reagents such as antibodies. In thisstudy, we applied the IMS technique to formalin-fixed paraffin-embedded (FFPE) samples to
identify a substance(s) responsible for the intestinal obstruction caused by an unidentified foreign
body. In advance of IMS analysis, some pre-treatments were applied. After deparaffinization ofsections, samples were subjected to enzyme digestion. The sections co-crystallized with matrix
were desorbed and ionized by a laser pulse with scanning. With IMS Convolution software which
we developed, we could automatically extract meaningful signals from IMS datasets. The
representative peak values were m/z 1013, 1175, 1337, 1499, 1661, 1823, and 1985. Thus it was
revealed that the material was polymer with 162 Da unit size, calculated from the even intervals.
In comparison with mass spectra of the histopathological specimen and authentic materials, themain component coincided with amylopectin, rather than amylose. Tandem MS (MS/MS) analysis
proved that the main component were oligosaccharides. Finally, we confirmed the identification ofamylopectin by staining with periodic acid-Schiff (PAS) and iodine. These results for the first timeshow the advantages of MALDI-IMS in combination with enzyme digestion for direct analysis ofoligosaccharides as a major component of histopathological samples.
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Conclusion:
With direct MALDI-TOF MS imaging, we identified mochi as the obstructing material in asmall intestinal obstruction. The results in this study indicate that IMS is a very rapid and accurate
technique with high sensitivity for direct analysis for histopathological samples. This is the firstcase of identification of oligosaccharides as a main component of histopathological samples using
direct MS analyses. MALDI-TOF MS in combination with enzyme digestion will be a support for
the histopathological study (Yamada et al., Anal. Bioanal. Chem.402, 1921-1930, 2012).
質量顕微鏡法を用いて腸閉塞の原因となった異物の成分を同定した。
1)質量顕微鏡法を用い外科手術で摘出された異物の病理組織標本から位置情報と質量ス
ペクトルを同時取得する。
食餌性異物による腸閉塞・穿孔性腹膜炎を併発した症例に対して、食餌性異物を含む病
理標本パラフィンブロックから 4μm 厚の切片を作製し、脱パラフィン処理後、適当な消化酵素を用い、標本中の分子を切断し、前処理を標本上で行った。前処理したサン
プルを質量顕微鏡法により解析した。
2)質量顕微鏡法で画像化された分子種の同定を行う。
質量顕微鏡法で得られた情報から物質の成分を推定した。ポリマーのユニット構造を予想したところ多糖類であることが予想された。更に、多段階質量分析 MS/MS 測定を用いて病理組織標本を解析した結果、グルコースをユニットとするアミロペクチンであることが判明した。これは餅の成分であることが知られている。
3)各種の特殊病理染色法を用い検証する。
病理検査で行われている各種の特殊染色法で組織標本の染色を行った。異物の病理標本を作製し、特殊染色を施した結果、糖原か炭水化物の可能性を示唆する PAS 染色で陽性の結果を得た。S-100 蛋白染色による腫瘍検出、Kossa 染色によるカルシウム検出、及びDylon・Congo red でのアミロイド検出はすべて陰性であった。また CD68 染色も陰性であり、生体内での異物反応は認めなかった。
以上の結果より、腸閉塞の原因物質が餅であることが明らかとなった。

2. List of Publications
Original Articles
1. Yamada, M., Yao, I., Hayasaka, T., Ushijima, M., Matsuura, M., Takada, H., Shikata, N.,
Setou, M., Kwon, A-H and Ito, S. Identification of oligosaccharides from histopathologicalsections by MALDI imaging mass spectrometry. Anal. Bioanal. Chem. 402, 1921-1930, 2012.

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Search and Identification of head and neck cancer-related factors by imaging
Title of Projectmass spectrometry
質量顕微鏡による頭頸部がん関連因子の探索・同定

P.I.

Koichi Tomoda, 友田幸一

E-mailtomodak@hirakata.kmu.ac.jp

Affiliation

Otolaryngology, 耳鼻咽喉学講座

Collaborators

岡崎はるか、神田靖士、大岡久司

Keywords

ABCG2, auditory center, Sca1, side population cell, stem cell

1. SUMMARY OF RESEARCH RESULTS
To investigate the keys of treatments for the deaf patients that have auditory damages, we tried toisolate stem/progenitor cells from the auditory pathway and analyzed the cell characterization andgene expressions. We assumed that it was possible to purify stem cells as side population (SP) cells,
cochlear nuclei and inferior collicullus cells were isolated from 2-4 weeks age of mice and stainedwith Hoechst 33342. We analyzed the gene expression of SP cells by microarray techniques and
RT-PCR and the specific markers of stem/progenitor cells, such as ABCG2 and Sca1, wereover-expressed in SP cells compared with main population (MP) cells. Spheroid formation was notdetected when the SP cells from cochlear nuclei and inferior collicullus were grown in serum-free,
non-adherent culture.
①マウス蝸牛神経核・下丘において SP 細胞、MP 細胞の遺伝子発現を DNA Microarray にて検討した結果、MP 細胞に比べて SP 細胞において BCRP1/ABCG2 などの遺伝子発現が強くみられた。この結果から SP 分画は幹細胞に富む分画であることが示唆された。次に、Mictoarray の結果より SP 細胞において強発現していた Sca1、ABCG2 の発現は、SP 細胞において MP 細胞より強い事が確認された。
②幹細胞の自己複製能を検討するため、C57BL/6 GFP+マウス、C57BL/6 WT マウスを用いて SP 細 胞 を neural stem cell medium (containing neurobasal-A medium, GlutaMAX
suplement,B27 suplement, Penicillin/Streptomyscin, EGF and FGF)を用いて培養を行っているが、現在までに sphere の形成は確認できていない。
③現在実験に用いている C57BL/6 マウスが早期難聴を来すマウスであるためスイス系マウスなど早期難聴を来さないマウスとの比較検討も行っている。現在のところ、生後 1 週のBALB/c マウスの蝸牛神経核、下丘を単細胞に分離し、Hoechst 33342 にて染色後、FACS にて分析したところ全細胞中に SP 細胞を約 1％認めている。

2. List of Publications
Original Articles
1. Ooka, H., Kanda, S., Okazaki, H., Suzuki, H., Mishima, K., Saito, I., Yagi, M., Tomoda, K.,
Nishiyama, T. Characterization of side population (SP) cells in murine cochlear nucleus. Acta
Oto-Laryngologica, 2012 (in press)-97-Annual Report 研究成果報告書
Title of Project

Examination of application of IBM-BMT to intractable diseases of the nervous
system
難治性神経疾患に対する骨髄内骨髄移植の適用の検討

P.I.

Susumu Ikehara, 池原進

Affiliation

Stem Cell Disorders, 幹細胞異常症学（共同研究講座）

Collaborators

李銘、李清、石明Keywords

autoimmune disease, IBM-BMT, thymus

E-mailikehara@takii.kmu.ac.jp

1. SUMMARY OF RESEARCH RESULTS
Our research clearly shows that currently incurable diseases with aging-associated symptoms arelinked to abnormalities of the immune system, and that both IBM-BMT and thymus transplantation
are effective interventions in the treatment of these diseases.
These diseases are also characterized by abnormalities in bone marrow mesenchymal stem cells,
but not hematopoietic stem cells.
We collaborated with the First Department of Medicine and Department of Orthopedic Surgery
for clinical application.
1) 研究の背景と目的
我々が開発した新しい骨髄内骨髄移植(IBM-BMT)の技術はヒト同種 BMT の主要な問題
点を解決する革新的技術であり、造血幹細胞の異常に基づく白血病や自己免疫疾患のみならず、間葉系幹細胞の異常に伴って発症する難治性の自己免疫疾患や加齢に伴って発症す
る種々の難病（アルツハイマーや肺気腫や骨粗鬆症等）の根治療法の開発につながる。難病のモデル動物の実験に基づき、いかなる難病が新技術で治療可能かを明らかにして、ヒトへの応用を目的とする。
2) 結果
加齢に伴って発症する難病には、免疫の異常が関与しており、骨髄内骨髄移植だけでな
く胸腺の移植の併用が重要であることを明らかにした（文献②，⑤，⑦，⑨）。
さらに、加齢に伴って発症する疾患は、骨髄の造血幹細胞ではなくて、間葉系の幹細胞の異常に起因することを見出した（文献⑦）。
新しい骨髄移植のヒトへの応用を目指して、第一内科と整形外科との共同研究を開始している。

2. List of Publications
Original Articles
1. 池原 進革新的移植方法 ―灌流法＋骨髄内骨髄移植法
医学のあゆみ「造血幹細胞移植の最新動向：造血幹細胞移植のトピックス」 240 (5):465-469, 2012.
2. Zhang Y, Hosaka N, Cui Y, Shi M, and Ikehara S.
Effects of allogeneic hematopoietic stem cell transplantation plus thymus transplantation on
malignant tumors: Comparison between fetal, newborn, and adult mice.
Stem Cells Dev. 20: 599-607, 2011.
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3. Oe K, Kushida T, Okamoto N, Umeda M, Nakamura T, Ikehara S, and Iida H.
New strategies for anterior cruciate ligament partial rupture using bone marrow transplantationin rats. Stem Cells Dev. 20: 671-679, 2011.
4. Shi M, Lian Z, Zhang Y, Yanai S, Shima C, Imai Y, and Ikehara S.
Combination of intra-bone marrow-bone marrow transplantation and subcutaneous donor
splenocyte injection diminishes risk of GVHD and enhances survival rate.
Stem Cells Dev. 20: 759-768, 2011.
5. Ikehara S.
Thymus transplantation for treatment of cancer: lessons from murine models.
Expert Rev. Clin. Immunol. 7: 205-211, 2011.
6. Mori S, Fujita S, Yamamoto Y, Li M, Fukuhara S, Nomura S, and Ikehara S.
Perfusion method for bone marrow cell collection in poor mobilizer lymphoma patient.
Int. J. Hematol. 93: 822-824, 2011.
7. Ikehara S.
A novel BMT technique for treatment of various currently intractable diseases.
Best Pract. Res. Clin. Haematol. 24: 477-483, 2011.
8. Hoshino S, Kurishima A, Inaba M, Ando Y, Fukui T, Uchida K, Nishio A, Iwai H, Yokoi T, Ito
T, Ishii S, Shimada A, Li M, Okazaki K, and Ikehara S.
Amelioration of 2,4,6-trinitrobenzene sulfonic acid-induced colitis in mice byimmunoregulatory dendritic cells.
J. Gastroenterol. In press. 46: 1368-1381, 2011.
9. Zhang Y, Hosaka N, Cui Y, Shi M, Li M, Li Q, and Ikehara S.
Effects of intra-bone marrow-bone marrow transplantation plus adult thymus transplantation on
survival of mice bearing leukemia.
Stem Cells Dev. In press.
Proceedings
1. Susumu Ikehara.
Autoimmune diseases as stem cell disorders: Rationale for normal stem cell transplantation for
their treatment. The 5th Autoimmunity Congress Asia (ACA 2011).招聘講演 November
17-19, 2011 (Singapore)
2. LI Ming, SHI Ming, IKEHARA Susumu．
骨髄と胸腺の同時移植による２型糖尿病の治療／Treatment of type 2 diabetes
mellitus in db/db mice by intra-bone marrow-bone marrow transplantation plus thymustransplantation.第 40 回日本免疫学会学術集会 平成 23 年 11 月 27 日～29 日（千葉）3. 保坂直樹，崔雲澤，石明，李銘，李清，高橋伯夫，池原進．
白血病担癌マウスにおける成体胸腺移植を併用した IBM-BMT の効果／Effects of
IBM-BMT plus adult thymus transplantation on mice bearing leukemia.
第 40 回日本免疫学会学術集会 平成 23 年 11 月 27 日～29 日（千葉）-99--100-

Division of
Metabolic Disorders

代謝部門

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Annual Report 研究成果報告書
Title of Project

Study of fibril formation in the extracellular matrix
細胞外マトリックス線維形成の加齢変化に関する研究

P.I.

Tomoya Akama, 赤間智也

E-mailakamat@takii.kmu.ac.jp

Affiliation

Neuropharmacology, 神経病態薬理学（薬理学講座）

Collaborators

赤間智也、東淳子、野田和男、井上唯史、木村あすな

Keywords

elastic fibers, extracellular matrix, gene-engineered mice

1. SUMMARY OF RESEARCH RESULTS
Tissue elasticity depends on formation of elastic fibers in the extracellular matrix of the tissue.
Malformation or degradation of elastic fiber results in stiff, inflexible tissues, such as tortuousartery, lung emphysema and wrinkled skins. We are studying on formation of elastic fibers in vivousing gene knockout mice, and have found several key molecules for the fiber organization.
Latent TGF binding protein 4 (LTBP4) is one of the LTBP family proteins, which bind to
TGF to be inactivated and store it in the extracellular matrix until additional signals such asproteases release TGF. We found that amount of LTBP4 protein was greatly decreased in skinsamples of an old human donor than that of a young donor, suggesting LTBP4 protein is involvedin elastic fiber formation in vivo. We then analyzed LTBP4-mutant mice and found that thehomozygous mutant mice had stiff, tortuous artery with inelastic property, strongly indicating
important functions of LTBP4 on elastic fiber formation in vivo. By fiber formation assay on
primary cultured human dermal fibroblasts, we knocked down gene expression of LTBP4 in the
fibroblast cells by siRNA and found that loss of LTBP4 expression completely diminishedformation of elastic fiber meshwork in the extracellular matrix formed on the surface of thecultured cells. Conversely, we included recombinant LTBP4 protein in the culture medium offibroblast cells with suppressed LTBP4 expression and found restoration of elastic fiber meshwork
formation in the cells. Since production of elastic fiber meshwork was increased by addition of
LTBP4 protein in a dose-dependent manner, we concluded that LTBP4 protein is a rate-limiting
factor for elastic fiber formation in the cultured cells.
Because LTBP4 is a family protein of LTBPs, which are involved in TGF signaling pathway,
there was a speculation that enhancing effect of LTBP4 for elastic fiber formation is due to TGFmolecules attached to recombinant LTBP4 protein. To clarify molecular function of LTBP4
protein on elastic fiber formation, we prepared recombinant LTBP4 protein that holds no active
TGF molecule and tested this TGF-free LTBP4 protein for elastic fiber formation assay on
cultured cells. Surprisingly, TGF-free LTBP4 showed promoting effect for elastic fiber formation
as strong as normally prepared recombinant LTBP4 protein that held the minimal amount of TGFmolecules, indicating promoting effect of LTBP4 protein for elastic fiber formation is anindependent function from TGF signaling pathway. By solid-phase binding assay, we found that
LTBP4 protein binds to tropoelastin molecule in presence of fibulin-5, which is anotherextracellular matrix protein known to be involved in elastic fiber formation. From these findings,
We hypothesized that LTBP4 works as a scaffold of the elastin molecules on microfibrils made offibrillins, and tropoelastin/fibulin-5 complex localize on microfibrils by binding to LTBP4 as amarker. Accumulated elastins are subsequently crosslinked and matured to be functional elastic
fibers. We discovered that LTBP4 is a key molecule at the early step of elastic fiber formation.
細胞外マトリックスの一つである弾性線維は固く丈夫なコラーゲン線維とは異なり、柔らかく伸び縮みする線維である。弾性線維は肺や大動脈、皮膚といった伸縮性のある組織の細胞外マトリックスに存在し、機能している。我々は弾性線維形成機構の解明に向けて研究を進めており、遺伝子改変マウスや培養細胞系を用いて弾性線維形成に必須な遺伝子群とその役割の解析を行なっている。
弾性線維の主な構成成分はエラスチン重合体であり、細胞によって合成、分泌されたエ
ラスチン単量体は同じく細胞外マトリックスの構成成分であるフィブリリン 1・2 からなる微小線維上に沈着し、リシルオキシダーゼ群の酵素活性により酸化的に架橋され、最終-102-

産物のエラスチン重合体となり弾性線維として機能する。しかしながらこれらの弾性線維構成成分を培養細胞中に発現させても弾性線維が合成されることはなく、上記タンパク質
以外にも弾性線維形成に関わる未知のタンパク質の存在が予想されていた。
我々はこれまで弾性線維合成に必須である細胞外マトリックスタンパク質を発見し、その遺伝子変異マウスが弾性線維形成にかかわることを示してきた。LTBP4 (latent-TGF
binding protein 4)は TGFb を不活性化状態に維持して細胞外マトリックス内に留め置くタンパク質である LTBP ファミリーの一つであるが、我々は弾性が減少した老人の皮膚組織には若年者の皮膚組織に比べ LTBP4 の存在量が大きく減少していること、LTBP4 欠損マ
ウスが大動脈の硬化を示すことを発見し、LTBP4 が弾性線維構築に重要な機能を担っていることを示した。さらには精製 LTBP4 タンパク質をヒト皮膚線維芽細胞の培養液に加えることにより、培養細胞上に弾性線維様の網目状線維構造を形成させることができ、この線維構造は LTBP4 の添加量に比例して増加したことから、LTBP4 の発現量が弾性線維形成の律速となっていることが示唆された。更なる解析にて LTBP4 はエラスチン/fibulin-5複合体を微小線維上に誘導する機能を有していることがわかり、LTBP4 の弾性線維形成における新たな役割が明らかとなった。

2. List of Publications
Original Articles
1. Otani, H., Yoshioka, K., Nishikawa, H., Inagaki, C., Nakamura, T., Involvement of Protein
Kinase C and RhoA in Protease-Activated Receptor 1-Mediated F-Actin Reorganization andCell Growth in Rat Cardiomyocytes. J. Pharmacol. Sci. 115 (2011) 135-143.
2. Ohara, H., Akatsuka, S., Nagai, H., Liu, Y., Jiang, L., Okazaki, Y., Yamashita, Y., Nakamura,T., Toyokuni, S., Stage-specific roles of fibulin-5 during oxidative stress-induced renal
carcinogenesis in rats. Free Radic. Res. 45 (2011) 211-220.
3. Tamura, N., Sugihara, K., Akama, T.O., Fukuda, M.N., Trophinin-mediated cell adhesion
induces apoptosis of human endometrial epithelal cells through PKC-delta. Cell Cycle10(2011) 135-143.
4. Parfitt, J.G., Pinali, C., Akama, T.O., Young, R.D., Nishida, K., Quantock, A.J., Knupp, C.,
Electron tomography reveals multiple self-association of chondroitin sulfate/dermatan sulfate
proteoglycans in Chst5-null mouse corneas. J. Struct. Biol.174 (2011) 536-541.
5. Hatakeyama, S., Sugihara, K., Shibata, K.T., Nakayama, J., Akama, T.O., Tamura, N., Wong,
S-M., Bobkov, A.A., Takano, Y., Ohyama, C., Fukuda, M., Fukuda, M.N., Targeted drugdelivery to tumor vasculature by a carbohydrate mimetic peptide. Proc., Natl. Acad. Sci.
USA.108 (2011) 19587-19592.
6. Shibata, T.K., Matsumura, F. Wang, P., Yu, S., Choo, C.C., Khoo, K.H., Kitayama, K.,
Akama, T.O., Sugihara, K., Kanayama, N., Kojima-Aikawa, K., Seeberger, P.H., Fukuda, M.,
Suzuki, A., Aoki, D., Fukuda, M.N., Identification of mono- and di-sulfatedN-acetyllactosaminyl oligosaccharide structures as epitopes specifically recognized byhumanized monoclonal antibody HMOCC-1 raised against ovarian cancer. J. Biol. Chem.
287 (2012) 6592-6602.
Proceedings
1. Nakamura, T., The essential role of LTBP4 in elastic fiber assembly. Invited talk at Gordon
Research Conference on Elastin and Elastic Fiber, Biddeford, U.S.A., July 24-28, 2011
2. Noda, K., Takagi, K., Horiguchi, M., Inoue, T., Akama, T.O., Melchner, v. H., Hyytiainenn,
M., Suzuki, S., Nakamura, T., The essential role for LTBP4 in elastic fiber assembly. Poster
presentation at Gordon Research Conference on Elastin and Elastic Fiber, Biddeford, U.S.A.,
July 24-28, 2011 and selected oral presentation at Gordon Research Seminar on Elastin and-103-

Elastic Fiber, Biddeford, U.S.A., July 23-24, 2011
3. Inoue, T., Ohbayashi, T., Horiguchi, M., Kusumoto, K., Hirai, M., Nakamura, T., The role of
LTBP-2 in eye development. Poster at Gordon Research Conference on Elastin and Elastic
Fiber, Biddeford, U.S.A., July 24-28, 2011 and selected oral presentation at Gordon Research
Seminar on Elastin and Elastic Fiber, Biddeford, U.S.A., July 23-24, 2011
4. Akama, T. O., Fukuda, M., Fukuda, M.N., Nakamura, T., Distinct roles ofb1,3-N-acetylglucosaminyltransferases and carbohydrate 6-O sulfotransferases on corneal
keratan sulfate production, 21st International Symposium on Glycoconjugates, Vienna,
Austria, August 21-26, 2011
5. Akama, T. O., Fukuda, M., Fukuda, M.N., Nakamura, T., Study of corneal keratan sulfate
biosynthesis in vivo using sulfotransferase gene knockout mice, Annual Conference of theSociety for Glycobiology, Seattle, U.S.A., November 9-11, 2011
Patents
1. 欧州特許登録（EPC） 出願番号 05720545.2
件名：切断型DANCE、DANCE複合体、及びこれらを用いる方法
登録日：2011. 10. 19
登録番号：1762610
権利者：学校法人関西医科大学
発明者：中邨智之 平井希俊

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Annual Report 研究成果報告書
Title of Project

Analysis of signal transduction molecules involved in proliferation,
development and stemness
がん幹細胞の機能・分化に関わるシグナル分子の解明

P.I.

Satoshi Matsuda, 松田達志

Affiliation

Cell Signaling, 生体情報部門

Collaborators

大谷真志

Keywords

cell proliferation, mTOR, PI3K

E-mailmatsudsa@takii.kmu.ac.jp

1. SUMMARY OF RESEARCH RESULTS
Members of the phosphoinositide 3-kinase (PI3K) family are important for the differentiation,
proliferation, and survival of a variety of cells, including immune cells. The basic activity of these
enzymes is to phosphorylate the third position of the hydroxyl group in the inositol ring ofphosphatidylinositol. There are four subclasses of the PI3K family; among these, class IA PI3K,
composed of a catalytic subunit (p110, , or ) and a regulatory subunit (p85, p55, p50,
p85, or p55), works downstream of receptors such as T cell receptor (TCR) and CD28 in T cells
whose signaling pathways involve protein tyrosine kinases and Ras. In many cell types, PI3K
signals through mammalian target of rapamycin complex1 (mTORC1) consisting of mTOR,
raptor, and mLST8. For instance, it has been shown that PI3K-mTORC1 axis regulates thenuclear translocation of IRF7 for the production of type I interferon (IFN) in plasmacytoid
dendritic cells. Production of IL-12 and IL-10 in conventional dendritic cells is also regulated bymolecules downstream of PI3K including mTORC1. In CD4+ T cells, the PI3K-Akt-mTORC1
axis contributes to the activation, survival, and proliferation upon stimulation through TCR andCD28. In collaboration with Koyasu Laboratory in Keio University School of Medicine, we
found that the suppression of this axis by deletion of p85 or PI3K/mTORC1 inhibitors as well
as T cell-specific deletion of raptor, an essential component of mTORC1, impaired Th17
differentiation in vitro and in vivo in a S6K1/2-dependent fashion.
Inhibition of
PI3K-Akt-mTORC1-S6K1 axis impaired the downregulation of Gfi1, a negative regulator of Th17
differentiation. We further found that S6K2, a nuclear counterpart of S6K1, was induced by the
PI3K-Akt-mTORC1 axis, bound ROR , and carried ROR to the nucleus. These results point
toward a pivotal role of PI3K-Akt-mTORC1-S6K1/2 axis in Th17 differentiation.
PI3K-mTOR 経路は細胞の増殖・分化・生存を制御する細胞内シグナル伝達経路であり、種々のがん組織においてその異常が報告されている。本研究では、PI3K-mTOR 経路に関わる各種分子の遺伝子改変マウスを対象に、これらシグナル伝達経路の活性の増減が各種
血球系細胞の分化・増殖過程に及ぼす影響の解析を試みた。
本年度は、クラス IA PI3K の制御サブユニットである p85を欠失したマウス（以下、
p85-KO マウス）、クラス IB PI3K の触媒サブユニットである p110を欠失したマウス（以下、p110-KO マウス）、両者を共に欠失した p85/p110二重欠損マウス（以下、DKO）
を用いて解析を行った。対象として、T 細胞、B 細胞、マスト細胞、樹状細胞、マクロフ
ァージの分化に関して解析を行ったところ、p85-KO マウスにおいて B 細胞ならびに腸管マスト細胞の分化異常が確認されると同時に、ごく一部の未熟胸腺 T 細胞の分化過程にも異常が確認された。p110-KO マウスにおいては、胸腺 T 細胞の分化過程に部分的な異常が観察される一方、B 細胞やマスト細胞の分化に目立った異常は認められなかった。
DKO においては、単独の PI3K の欠失では認められない胸腺 T 細胞数の著しい減少と、分化異常が観察された。すなわち、クラス IA PI3K とクラス IB PI3K 経路が、胸腺 T 細胞の分化過程において、互いに冗長なシグナル伝達経路を構成していることが強く示唆された。興味深いことに、PI3K の下流で機能する mTOR 経路の中でも、mTORC1 シグナルに-105-

必須の Raptor 分子を T 細胞系列特異的に欠失させたマウスにおいては、そのような胸腺細胞数の減少は認められず、末梢の T 細胞機能に部分的な異常が観察されるにみであった。一方、マスト細胞の細胞数に関して、DKO と p85-KO マウスの間で著名な差は認められなかったものの、in vitro におけるマスト細胞の分化誘導系を用いたところ、DKO マ
ウス由来の血球系幹細胞を用いた際には、明らかな分化の遅れが観察された。すなわち、
クラス IA PI3K 経路が腸管におけるマスト細胞の分化・生存に必須な役割を果たすと同時に、血球系幹細胞からの効率的なマスト細胞分化誘導にクラス IA・クラス IB PI3K 経路が協調的に関わっていることが強く示唆された。

2. List of Publications
Original Articles
1. Kurebayashi, Y., Nagai, S., Ikejiri, A., Ohtani, M., Ichiyama, K., Baba, Y., Yamada, T., Egami,S., Hoshii, T., Hirao, A., Matsuda, S., and Koyasu, S. PI3K-Akt-mTORC1-S6K1/2 axis
controls Th17 differentiation by regulating Gfi-1 expression and nuclear translocation of
ROR Cell Reports (2012) in press
2. Yokoyama, T., Matsuda, S., Takae, Y., Wada, N., Nishikawa, T., Amagai, M., and Koyasu, S.
Antigen-independent development of Foxp3+ regulatory T cells suppressing autoantibody
production in experimental pemphigus vulgaris. Int. Immunol・23・2011・365-373.
Proceedings
1. Yamana, A., Yamashita, A., Suzuki, M., Matsui, Y., Watanabe, T., and Matsuda, S. Forced
expression of a constitutively active form of p110δ results in decrease of double positive
thymocytes during thymocyte development. 第 34 回分子生物学会総会・横浜・2011 年
12 月
2. Matsuda, S. and Ohatni, M. Role of the mTORC1 signaling pathway in T cell development.
第 40 回日本免疫学会学術集会・幕張・2011 年 11 月
3. Ohatni, M. and Matsuda, S. Role of mTORC1 in immune function of dendritic cells. 第 40回日本免疫学会学術集会・幕張・2011 年 11 月
4. Nagai, S., Kurebayashi, Y., Baba, Y., Matsuda, S., and Koyasu, S. PI3K-Akt-mTORC1 axis
controls Th17 differentiation by regulation of Gfi-1 and RORγt. 第 40 回日本免疫学会学術集会・幕張・2011 年 11 月
5. Fujii, H., Matsuda, S., Ohtani, M., and Koyasu, S. Role of class IA PI3K in regulatory T cellfunction. 第 40 回日本免疫学会学術集会・幕張・2011 年 11 月
6. 松田達志、大谷真志、星居孝之、平尾敦 Ｔ細胞分化過程における mTORC1 シグナルの役割 第 21 回 Kyoto T Cell Conference・京都・2011 年 6 月
7. 藤猪英樹、小安重夫、松田達志 抑制性Ｔ細胞機能における PI3K の役割
Kyoto T Cell Conference・京都・2011 年 6 月-106-

第 21 回Annual Report 研究成果報告書

Title of Project

Development of novel therapeutic strategy and elucidation of functional basis
for intractable diseases by human dendritic cell research
ヒト樹状細胞によるさまざまな病態関連応答の解明と新たな難病治療戦略の開発

P.I.

Tomoki Ito, 伊藤量基

Affiliation

Internal Medicine 1, 内科学第一講座

Collaborators

野村昌作、安室秀樹、宮本理恵、杉本博是、中西孝尚

Keywords

autoimmune disease, dendritic cells, interferon-, platelet, statin, TSLP

E-mailitot@takii.kmu.ac.jp

1. SUMMARY OF RESEARCH RESULTS
1. Systemic lupus erythematosus (SLE) is a severe and intractable autoimmune diseasecharacterized by chronic activation of plasmacytoid dendritic cells (pDCs) and production of
autoantibodies against nuclear self-antigens. Neutrophils are also implicated in disease
pathogenesis; however, the mechanisms involved are unknown. Here, we identified in the sera of
SLE patients immunogenic complexes composed of neutrophil-derived antimicrobial peptides andself-DNA. These complexes were produced by activated neutrophils in the form of neutrophil
extracellular traps (NETs) and efficiently triggered innate pDC activation via TLR9. Circulating
neutrophils from SLE patients released more NETs than those from healthy donors; this was
further stimulated by the antimicrobial autoantibodies.
In addition, our study identified that both BAY11 (agent for NFkB inhibitor) and statins(HMG-CoA reductase inhibitors) have the ability to inhibit nuclear translocation of IRF7 andIFN- production in human pDCs. We also found that these agents inhibited both in vitro IFN-
production induced by the SLE serum and the in vivo serum IFN- level induced by injecting
mice with TLR ligand. These findings suggest that BAY11 and statins have the therapeuticpotential to attenuate the IFN environment by regulating the pDC function and thus provide thenovel foundation for the development of an effective immunotherapeutic strategy against
autoimmune disorders such as SLE.
2. TSLP strongly activates human DCs and then induces robust expansion of not only naïve CD4+
T cells but also naïve CD8+ T cells. Based on these findings, we hypothesized TSLP is a good
adjuvant of expansion of CTL response in DC-mediated immune process by enhancing the effectof BCG. Although BCG-stimulated DCs induced CD8+ T cell production of of IFN- but notstrong cell expansion, TSLP-stimulated DCs induced robust CD8+ T cell expansion but not highquantities of IFN- production. We here found that DCs stimulated with both BCG and TSLP
induced robust expansion of CD8+ T cells that produce a large amount of IFN- with a potent
cytolytic activity related to the expression of granzyme B and TIA-1. Our data suggest that TSLP
is potent adjuvant in both quantitatively and qualitatively enhancing the CTL effect by BCG
immunotherapy through DC activation, and therefore provide a novel strategy for antitumor
immune-based therapy.
3. We newly demonstrated that TRAP (thrombin receptor agonist peptide)-activated platelets
expressed RANKL, which promoted the CD86 expression on DCs. We further clarified that theactivated platelets enhanced the production of CCL17 induced by the DC stimulated with TSLP.
Thus, platelets have the ability to enhance the DC-mediated Th2 response and may contribute to
the allergic inflammation. In conclusion, our study may provide new insights of platelet function
into the possible mechanism of allergic response that stems from DCs.
1. 近年、様々な炎症性疾患の病態発症･進展に、免疫システムの中枢に位置する樹状細胞（dendritic cell:DC）が重要な働きをすることが示唆されている。
自己免疫疾患は、本来抑制されるはずの自己成分に対する免疫応答に基づく病態の基盤である。特に、全身性エリテマトーデス (SLE)などの自己免疫疾患においては、樹状細胞の一亜群（形質細胞様樹状細胞 plasmacytoid DC:pDC）が自己核酸に応答して、I 型インター-107-

フェロン（IFN）産生を誘導することが、病態に関与していることが示唆されている。しかしながら、その詳細なメカニズム、すなわち自己免疫疾患では自己核酸に対する制御が如何に破綻し、IFN 産生を誘導しているのか、という点が不明であった。我々はその病態発症のメカニズム、免疫破綻の機序を解明し、さらに SLE に対して、pDC と I 型 IFN 産生を標的とした新たな治療戦略としての薬剤を検討した。
a.SLE では、自己の DNA の放出を、pDC が検知する際に、組織由来の抗菌ペプチド LL37
およびヒト好中球ペプチド human neutrophil peptides（HNP）が DNA と複合体を形成する
ことで安定化される。この現象は、好中球が障害を受ける時に、蜘蛛の巣状の形状を示す
核内 DNA を含んだ neutrophil extracellular traps (NETs)という成分を放出することで起きる。また、SLE 患者で認められる自己抗体が、この好中球による pDC の活性化において、助長する。これらの応答が SLE 患者では持続的に起きているため、慢性的に pDC からⅠ
型 IFN が産生され、SLE における病態の悪循環が形成されることを同定した。
b.この観点から、何らかの薬剤により、この pDC の持つ自己 DNA に対するⅠ型 IFN の産生機能を阻害することが出来るならば、SLE における病態形成の悪循環を断ち切る新たな治療戦略に繋がると期待される。我々は種々の薬剤をスクリーニングし、NF-κB 阻害剤
BAY11-7082 と高脂血症治療薬（HMG-CoA 還元酵素阻害剤：スタチン）を候補として考え、これらが pDC からの I 型 IFN 産生を阻害する活性をヒトならびにマウスにおいて見出した。この pDC 機能阻害活性は新規の知見であり、BAY11-7082 やスタチンのこの抑制的効果は、今後 SLE をはじめとする全身性自己免疫性疾患に対する新たな治療戦略に寄与すると考えられる。
2. 樹状細胞(DC)は抗原提示細胞として HLA 関連適応免疫応答において、重要な役割を果たす。CD4+T 細胞のみならず、CD8+T 細胞に対しても HLA-クラス I を介して抗原提示し naïve CD8+ T 細胞を、cytotoxic T 細胞 (CTL) に分化誘導することによって抗腫瘍免疫応答に寄与すると考えられている。一方、上皮由来サイトカインである胸腺
間質リンパ球増殖因子（TSLP）によって活性化した DC は、CD4+ T 細胞に対してのみな
らず、CD8+ T 細胞に対しても非常に強力な増殖誘導能を有することが明らかにされている。また従来より、Bacillus of Calmette and Guerin (BCG)は CTL 誘導能を示し、腫瘍免疫に有用なアジュバントと考えられている。今回我々は、TSLP と BCG を組み合わせることにより、TSLP 活性化 DC の持つ CD8+T 細胞増殖誘導能と BCG の持つ IFN-産生 CTL
分化誘導能とを併せ持つことが可能であり、これらが今後、抗腫瘍免疫療法の治療戦略において有力な免疫アジュバントであると考えられた樹状細胞免疫療法の可能性を検討した。
3.樹状細胞と活性型血小板の相互作用に関する研究を行っている。我々は新たに、活性化血小板が RANK リガンドを発現することを同定した。RANKL は TNF スーパーファミリーの一つである。RANKL 発現の活性化血小板によって、DC の持つ CD86 は発現増強し、さらに TSLP と同時に活性化血小板を添加することによって、DC から産生される CCL17は増加することも明らかにした。したがって、血小板は DC による Th2 免疫応答を増強し、アレルギー性の炎症を増幅させる可能性が示唆された。すなわち DC に由来するアレルギ
ー応答において、血小板による新たな増幅機序の可能性が考えられた。

2. List of Publications
Original Articles
1. Ito T, Arima K: Cellular and molecular mechanisms of TSLP function in human allergic
disorders--TSLP programs the “Th2 code” in dendritic cells--. Allergology International, 61,
35-43, 2012
2. Ito T. Plasmacytoid dendritic cells and type I interferon as therapeutic cellular and moleculartargets of autoimmune diseases
Cytometry Research 22:37-46,2012-108-

3. Nomura S, Fujita S, Nakanishi T, Yokoi T, Shimamoto K, Miyamoto R, Ito T: Platelet-derived
microparticles cause CD154-dependent activation of dendritic cells. Platelets,23,81-82,2011
4. Roberto Lande, Dipyaman Ganguly,Valeria Facchinetti,Loredana Frasca,Curdin Conrad, Josh
Gregorio, Stephan Meller, Georgios Chamilos, Rosalie Sebasigari, Valeria Riccieri, Roland
Bassett, Hideki Amuro, Shirou Fukuhara, Tomoki Ito, Yong-Jun Liu, Michel Gilliet
Neutrophils Activate Plasmacytoid Dendritic Cells by Releasing Self-DNA–Peptide
Complexes in Systemic Lupus Erythematosus
Sci Transl Med 2011: Vol. 3, p. 73ra19
5. 伊藤量基 形質細胞様樹状細胞をターゲットとした自己免疫疾患治療の新たな治療戦略, 臨床血液. 52.2011:p521-530
6. Hoshino S, Kurishima A, Inaba M, Ando Y, Fukui T, Uchida K, Nishio A, Iwai H, Yokoi T, Ito
T, Hasegawa-Ishii S, Shimada A, Li M, Okazaki K, Ikehara S.
Amelioration of 2,4,6-trinitrobenzene sulfonic acid-induced colitis in mice byimmunoregulatory dendritic cells.
J Gastroenterol. 2011 46:1368-81
7. Katashiba Y, Miyamoto R, Hyo A, Shimamoto K, Murakami N, Ogata M, Amakawa R, Inaba
M, Nomura S, Fukuhara S, Ito T.
Interferon-α and interleukin-12 are induced, respectively, by double-stranded DNA andsingle-stranded RNA in human myeloid dendritic cells.
Immunology 2011 :132 :165-173
8. Nomura S, Fujita S, Ozasa R, Nakanishi T, Miyaji M, Mori S, Ito T, Ishii K.
The correlation between platelet activation markers and HMGB1 in patients with disseminatedintravascular coagulation and hematologic malignancy.
Platelets. 2011, 22:396-397
9. Nomura S, Ozasa R, Nakanishi T, Fujita S, Miyaji M, Mori S, Yokoi T, Ito T, Ishii K.
Can recombinant thrombomodulin play a preventive role for veno-occlusive disease after
haematopoietic stem cell transplantation?
Thromb Haemost. 2011 105:p1118-1120.
10. Shosaku Nomura, Takahisa Nakanishi, Ryotaro Ozasa1, Shinya Fujita, Michihiko Miyaji,
Shinichiro Mori, Takashi Yokoi, Kazuyoshi Ishii, and Tomoki Ito
Dynamic role of cell-derived microparticles in hematopoietic stem cell transplantation
Current Trends in Immunology 2011 2011 12：21-27
Proceedings
1. 伊藤量基 全身性エリテマトーデスにおける重症度マーカーとしての血清 HMGB1 とLL37 日本血液学会総会・名古屋
2. 野村昌作 Preventive effects of recombinant thrombomodulin for veno-occlusive disease
after allogeneic stem cell transplantation 日本血液学会総会・名古屋
3. 野村昌作 Recombinant thrombomodulin may play a preventive role for veno-occlusive
disease after hematopoietic stem cell transplantation 国際血栓止血学会・京都-109-

Annual Report 研究成果報告書
Title of Project

Invention of cardiac regeneration therapy using molecular imaging techniques
分子イメージングによる心筋再生療法の開発

P.I.

Hajime Otani, 大谷肇

Affiliation

Internal Medicine 2, 内科学第二講座

Collaborators

宮坂陽子、岩崎真佳、藤田昌哲

Keywords

cardiac infarction, microRNA, regenerative medicine

E-mailotanih@takii.kmu.ac.jp

1. SUMMARY OF RESEARCH RESULTS
Background: MicroRNA-92a is a small noncoding RNA that has been shown to regulate
angiogenesis in a variety of normal tissues and tumors. We investigated whether controlled releaseof antagomir designed to inhibit microRNA-92a leads to enhanced blood vessel growth andfunctional recovery after myocardial infarction (MI) in rats.
Methods and Results: MI was created by ligation of the left coronary artery in rats. Gelatinhydrogel microspheres-incorporated patch impregnated with antagomir-92a was placed on thesurface of the infarct area of the left ventricle (LV) at the time of MI. There was no difference in
LV wall motion assessed by echocardiography between rats with the antagomir-92a patch and thepatch alone 1 hour after MI. However, myocardial thinning was inhibited, and LV wall motion
was improved 3 days after MI in rats with the antagomir-92a patch. Myocardial fibrosis in theinfarct area was reduced by the antagomir-92a patch 14 days after MI. Angiogenesis as evaluatedby the number of capillaries and small vessels within the infarct area was increased by the
antagomir-92a patch associated with enhanced cardiomyogenesis. The patch alone or the patch
impregnated with antagomir-Co had no effect on LV wall motion, myocardial fibrosis,
angiogenesis and cardiomyogenesis after MI.
Conclusions:Controlled

releaseofantagomir-92a

using

gelatinhydrogelmicrospheres-incorporated patch is an effective tool for cardiac regeneration therapy after MI.
目的: 本研究の目的は心筋梗塞ラットモデルにおいてマイクロ RNA 92a に拮抗するアン
タゴミア-92a 含有生体吸収性ゼラチンハイドロゲルパッチの心筋再生効果に関する検討を行うことである。心筋再生療法として血管増殖因子やケモカインを用いた分子治療が進められてきたが、単一のタンパクや遺伝子を標的とした分子治療には限界がある。そこで我々は心筋において血管新生を統合的に抑制していることが知られているマイクロ RNA
92a のアンタゴミアを用いて心筋再生を試みた。
方法: ラットの左冠動脈を結紮して心筋梗塞を作成し、梗塞巣にアンタゴミア 92a 含有生体吸収性ゼラチンハイドロゲルパッチ(Ant-92a パッチ)を逢着した。Bromodeoxyuridine(BrdU)を取り込んだ CD31+内皮細胞、c-kit+心筋幹細胞および α-actinin+心筋細胞数を測定して血管新生と心筋再生の程度を評価した。心筋梗塞 14 日後に超音波心エコーによって心機能を評価し、梗塞サイズを測定した。
結果：Ant-92a パッチはパッチ単独または Ant-92a のスクランブルオリゴヌクレオチドを含有したパッチと比較して梗塞巣において著明な血管新生を促した。それに伴い、心筋幹細胞の梗塞巣への集積と再生心筋細胞の有意な増加が認められた。その結果、梗塞サイズは有意に縮小し、左室機能は有意に改善した。
結論: Ant-92a パッチは心筋梗塞後の心筋再生療法として有用であることが示唆された。
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2. List of Publications
Proceedings
1. Fujita M, Otani H, Shimazu T, Yoshioka K, Okazaki T, Satoh D, Iwasaki M, Iwasaka T.
Controlled release of antagomir-92a using gelatin hydrogel microspheres-incorporated patch
promotes cardiac regeneration after myocardial infarction in rats.
第 75 回日本循環器学会学術集会. 横浜. 2011 年 8 月.
2. Fujita M, Otani H, Shimazu T, Yoshioka K, Iwasaki M, Iwasaka T, Tabata Y.
Inhibition of microRNA-92a enhances angiogenesis and cardiomyocyte regeneration throughintegrin 5-dependent accumulation of cardiac stem cells into the infarcted myocardium.
American Heart Association Scientific Sessions 2011. アメリカオーランド. 2011 年 11 月

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Annual Report 研究成果報告書
Title of Project

Molecular imaging in the development of carcinogenesis and tissue
regeneration in inflammatory diseases of the digestive organs.
炎症性消化器疾患の発癌と再生における分子イメージング解析

P.I.

Kazuichi Okazaki, 岡崎和一

Affiliation

Internal Medicine 3, 内科学第三講座

E-mailokazaki@hirakata.kmu.ac.jp

Collaborators
Keywords

inflammation, immunity in digestive organs, pSmad2/3L-Thr antibody,regeneration, stem cell

1. SUMMARY OF RESEARCH RESULTS
We studied Molecular imaging in the development of carcinogenesis and tissue regeneration in
inflammatory diseases of the digestive organs as follows.
1) In the upper alimentary tract, the gastric corpus and antrum are believed to contain epithelial
stem cells in the isthmus. However, the lack of useful markers has hindered studies of their origin.
We identified the significant expression of phosphorylation at specific linker threonine residues(pSmad2/3L-Thr) in specific epithelial cells of the murine stomach and have suggested these cells
to be epithelial stem cells.
2) In the lower alimentary tract, PIR-A/B(med) dendritic cells (DCs) serve as regulators by
inducing self-tolerance in the intestine and increase in number during the final stages ofinflammation, which suggested that PIR-A/B DCs can be used to treat colitis via an
IDO-dependent mechanism. In the development of colitic cancer, oncogenic Smad3 signaling,altered by chronic inflammation and eventually somatic mutations, promotes UC-associatedneoplastic progression by upregulating growth-related protein.
3) In the hepato-bilio-pancreas, patients with autoimmune pancreatitis (AIP) characteristicallyshow elevated serum levels of immunoglobulin G4 (IgG4) and abundant infiltration of
IgG4-positive plasmacytes in the involved organs such as the bile duct. We identified the
IgG4/IgG1 ratio in the liver may be a useful marker for differential diagnosis of IgG4-relatedsclerosing cholangitis (IgG4-SC) and primary sclerosing cholangitis (PSC). In IgG4-SC, abundant
infiltration of regulatory T cells (Tregs) may affect the switching of B cells to IgG4-producing
plasmacytes, and there is a possibility that the function of Tregs is different in IgG4-SC and otherliver diseases (PSC, AIH, and PBC).
In the development of hepatic malignancy,
fibro-carcinogenic signaling via phospho-Smad during progression of chronic viral liver diseases
such as viral hepatitis.
本研究は、消化器領域の炎症性疾患として H.pylori 感染胃・十二指腸疾患、炎症性腸疾
患（潰瘍性大腸炎やクローン病など）、膵炎（急性／慢性／自己免疫性膵炎）、肝炎（ウイ
ルス性、自己免疫性肝炎）を対象としている。研究期間において炎症と再生および発癌における炎症細胞と幹細胞の動態解析を各種抗体を用いたイメージング解析により、炎症と発癌ならびに臓器再生における分子機序を明らかにするとともに、これら疾患のバイオマ
ーカーの開発を行ってきており、具体的な成果は以下のとおりである。
1) 上部消化管：セリン残基 smad3 リンカー部リン酸化抗体を用いて、胃粘膜上皮幹細胞の候補を同定し、H.pylori 感染やアルコールによる傷害胃粘膜の再生過程における細胞動態とチオレドキシンによるレドックス機構による防御機構を明らかにした。
2)下部消化管：スレオニン残基 smad3 リンカー部リン酸化抗体を用いて、炎症性腸疾患における再生と癌化過程の分子機構を明らかにした。さらに、PIR-A/ B 抗体を用いて制御性樹状細胞を同定し、動物モデルを用いて炎症性腸疾患の新規治療法の開発の可能性を示し
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た。
3)肝胆膵：抗 FoxP3 抗体を用いて、自己免疫性肝疾患、IgG4 関連硬化性胆管炎、自己免疫性膵炎における ICOS 陽性制御性 T 細胞と IL-10 による病態制御機構の存在を明らかにした。さらにスレオニン残基 smad３リンカー部リン酸化抗体を用いて、B 型おより C 型
ウイルス性肝炎における発癌機構におけるバイオマーカーとしての意義を見いだした。

2. List of Publications
Original Articles
1. Hoshino S, Kurishima A, Inaba M, Ando Y, Fukui T, Uchida K, Nishio A, Iwai H, Yokoi T, Ito
T, Hasegawa-Ishii S, Shimada A, Li M, Okazaki K, Ikehara S. Amelioration of
2,4,6-trinitrobenzene sulfonic acid-induced colitis in mice by immunoregulatory dendritic
cells. J Gastroenterol. 201146(12):1368-81.
2. Kusuda T, Uchida K, Miyoshi H, Koyabu M, Satoi S, Takaoka M, Shikata N, Uemura Y,
Okazaki K. Involvement of inducible costimulator- and interleukin 10-positive regulatory T
cells in the development of igg4-related autoimmune pancreatitis. Pancreas. 201140(7):1120-30.
3. Watanabe T, Yamashita K, Fujikawa S, Sakurai T, Kudo M, Shiokawa M, Kodama Y, Uchida
K, Okazaki K, Chiba T. Activation of toll-like receptors and NOD-like receptors is involved inenhanced IgG4 responses in autoimmune pancreatitis. Arthritis Rheum. 2011 doi:
10.1002/art.33386
4. Nakase H, Tamaki H, Matsuura M, Chiba T, Okazaki K. Involvement of mycobacterium
avium subspecies paratuberculosis in TNF-α production from macrophage: possible link
between MAP and immune response in Crohn's disease. Inflamm Bowel Dis. 201117(11):E140-2. doi: 10.1002/ibd.21750.
5. Ikeura T, Takaoka M, Uchida K, Shimatani M, Miyoshi H, Kusuda T, Kurishima A, Fukui Y,
Sumimoto K, Satoi S, Ohe C, Uemura Y, Kwon AH, Okazaki K. Autoimmune pancreatitis
with histologically proven lymphoplasmacytic sclerosing pancreatitis with granulocytic
epithelial lesions. Intern Med. 2012;51(7):733-7
6. Nakajima A, Fukui T, Takahashi Y, Kishimoto M, Yamashina M, Nakayama S, Sakaguchi Y,
Yoshida K, Uchida K, Nishio A, Yodoi J,Okazaki K.
Attenuation of indomethacin-induced
gastric mucosal injury by prophylactic administration of sake yeast-derived thioredoxin. J
Gastroenterol. 2012 [Epub ahead of print]
Proceedings
1. Kazushige Uchida,Takeo Kusuda, Yutaku Sakaguchi, Katsunori Yoshida, Toshiro Fukui,
Akiyoshi Nishio, Kazuichi Okazaki. Possible role of ICOS and IL-10 Positive Regulatory T
Cells in the Development of IgG4-related Autoimmune Pancreatitis. American Pancreatic
Association Meeting. 2011/11. Chicago, USA.
2. Kazushige Uchida,Takeo Kusuda, Yutaku Sakaguchi, Katsunori Yoshida, Toshiro Fukui,
Akiyoshi Nishio, Kazuichi Okazaki. Possible role of ICOS and IL-10 Positive Regulatory T
Cells in the Development of IgG4-related Autoimmune Pancreatitis. American Pancreatic
Association Meeting. 2011/11. Chicago, USA.
3. Kazuichi Okazaki, Kimi Sumimoto,Tsukasa Ikeure,Kazushige Uchida,Makoto Takaoka. HOW
to recognize the mimickers of pancreas cancer in AIP? Japanese experience. Joint Meeting of
the 4th Asian- Oceanic Pancreas Association and 2011 Annual Congress of the Korean
Pancreatobiliary Association 2011/09. Jeju, Korea-113-

Annual Report 研究成果報告書

Title of Project

Clarification of skin and soft tissue aging and tissue regeneration andapplication in diagnosis and treatment using molecular imaging.
皮膚・軟部組織の加齢変化、組織再生における分子イメージングによる病態解明と診断治療への応用

P.I.

Kenji Kusumoto, 楠本健司

Affiliation

Plastic and Reconstructive Surgery, 形成外科学講座

Collaborators

鈴木健司、竹本剛司、堀尾修、日原正勝、覚道奈津子等 18 名

Keywords

blood vessel, bone, cartilage, cell, nerve, reconstruction, regeneration, skin,tissue, wound heeling

E-mailkusumoto@hirakata.kmu.ac.jp

1. SUMMARY OF RESEARCH RESULTS
In our research of 2011, bone regeneration, adipose-derived stem cells, and platelet-rich plasma
are in main studied.
Bone regeneration: Against critical sized calvarial bone defects of rats, bone morphogenetic
protein (BMP), hydroxyapatite (HAP), and the mixture (BMP/HAP) are implanted. Four weeks
after the implant, the regenerative bony areas are estimated radiologically and histologically using
molecular imaging and image analyzer. After subtraction technique of implanted HAP granules, inthe groups BMP/HAP, BMP, HAP, and control in this order, more massive regenerative bony
tissue are investigated. Within 4 weeks, the mixed BMP/HAP group showed the highest level ofbone induction.
Adipose-derived stem cells (ASCs): It is in vestigated that ASCs cytoskeletal changes inducedby the addition of C3 employing immunofluorescence staining, changes in alpha-smooth muscle
actin (α-SMA) gene expression, and the Rho-inhibitory effect. C3 significantly reduced stressfiber disruption and α-SMA expression 24hs after its addition at a concentration of 1 μg/ml, and it
also reduced the Rho activity level. While the correlation of the occurrence can be assumed, it
requires further examination to verify it. C3 may be an effective inhibitor of intracellular signaltransmission in ASCs cytoskeletal control involving Rho.
Platelet-rich Plasma (PRP):Autologous platelet-rich plasma provides multiple growth factors
after activation. A half-side test between the platelet-rich plasma (PRP)-treated and control(untreated) sides of a split-thickness skin graft donor site, and compared the number of days until
epithelialization and pain during gauze change. On day 13 after surgery, PRP promotes
epithelialization and angiogenesis of split-thickness skin graft donor sites.
On the other hand, in culture study α-SMA and gel contructure are shown with PRP. Then PRP
is thought that it contribute to wound healing promoting α-SMA producing thick epithelizationand scar contracture.
形成外科学講座では、創傷の生理的ならびに病的治癒過程を病態分子イメージングでとらえ、さらに種々の組織再生因子の負荷により病的創傷治癒が生理的創傷治癒に近づける
過程を病態分子イメージングとしてとらえることで、明らかな改善、治癒に関与するエビ
デンスを出すことを目指している。この病態分子イメージングにより創傷治癒、組織再生における基礎研究を治療学、再建学につなげる大きな役割を果たすものと考えている。
平成 23 年度は、骨再生、脂肪組織由来幹細胞、多血小板血漿（PRP）で、それぞれの成果を挙げた。
骨再生：ラット頭蓋骨の規格化した限界的骨欠損を作成し、骨形成タンパク（BMP）単独、あるいは hysroxyapatite（HAP）単独、両者併用で埋入した。この結果、骨欠損部の修復再生が生じるが、これをＸ線画像、組織学的所見での画像解析を行った。これにより、-114-

BMP が HAP より優れ、BMP+HAP 併用が最も骨欠損修復再生に効果的であることが明らかとなった。
脂肪組織由来幹細胞(ASCs)

細胞内に低分子量 G タンパク質 Rho が存在するが、exoenzyme C3 transferase が Rho inihibitor である。今回、C3 を加えることによる ASC
cytoskeleton の変化を観察した。C3 負荷によりストレスファイバーや α-SMA の発現も抑制された。C3 は ASCs の細胞内伝達の抑制因子である可能性が示唆された。
多血小板血漿(PRP) 均一採皮創で、PRP をハーフサイドで上皮化を比較検討した。術後１３日目に組織生検を行い PRP 群で、有意に表皮が厚く、血管数と αSMA が多かった。
PRP の有意なる創傷治癒促進効果と判断した。また、PRP は有意に αSMA の増加と瘢痕拘縮を生じることを明らかにした。

2. List of Publications
Original Articles
1. Notodiharjo FZ, Kakudo N, Kushida S, Suzuki K, Kusumoto K: Bone regeneration with
BMP-2 and hydroxyapatite in critical-size calvarial defects in rats. Journal of
cranio-maxillo-facial surgery. 40:287-291,2012.
2. Kakudo N, Kushida S, Minakata T, Suzuki K, Kusumoto K.: Platelet-rich plasma promotes
epithelialization and angiogenesis in a splitthickness skin graft donor site. Medical molecularmorphology 44(4):233-236,2011.
3. Kakudo N, Kushida S, Suzuki K, Matsumoto N, Kusumoto K.: The effect of C3 transferase on
human adipose-derived stem cells. Human cell 24(4):165-169,2011.
4. Kushida S, Kakudo N, Suzuki K, Kusumoto K.:Effects of platelet-rich plasma on proliferationand myofibroblasttic differentiation in human dermal fibroblasts. Ann Plast Surg in press
Proceedings
1. Kusumoto Kenji, Suzuki Kenji, Fukuda Satoshi, Miyake Yoshikazu, Kakudo Natsuko, Tanaka
Yoshihito, Sasao Takashi, Kushida Satoshi, Katsube Motoki, Valentin Priscilla, Ogura
Tsunetaka: Autologous Platelet-rich Plasma(PRP) and the Applications for wrinkle care andulcer treatment. The 21st Japan-China Joint Congress on Plastic Surgery, Fukuoka city,
2011/11-115-

Annual Report 研究成果報告書
Title of Project

Cellular function imaging by x-ray microscopy
軟Ｘ線顕微鏡による細胞内機能イメージング

P.I.

Hiroshi Kihara, 木原裕E-mailkihara@makino.kmu.ac.jp

Affiliation

Physics, 物理学講座

Collaborators

楠本（竹本）邦子、新庄正路、松村義隆、市村薫

Keywords

carboxysome, cryogenic observation, folding, protein, soft X-ray microscopy,
X-ray slution scattering

1. SUMMARY OF RESEARCH RESULTS
1. X-ray microscopy imaging(1) X-ray microscopic examinations of carboxysome
It is known that Leptolyngbya tenuis (Phormidium tenue) is a blue-green alga producing musty
odor in Lake Biwa. L. tenuis was cultured from the water of Lake Biwa and the cells have been
observed by soft x-ray microscope. Cell suspension without chemical fixation or staining was
dropped onto a polyimide thin film and air-dried. Two wavelengths, 2.0 nm and 2.4 nm (below
and above the wavelength of K-absorption edge of oxygen, 2.28 nm), were used for the x-ray
observation. Granules were also confirmed by 2.0 nm observations clearly. The diameter of
granules was about 500 nm. However the granules were not observed by 2.4 nm observations.
These results show that oxygen is most important constituent element for the granule. To identify
the granules, the transmission electron microscope (TEM) and the indirect fluorescent-antibody
technique (IFA) were done. IFA and TEM are effective methods to study of phytoplankton. By
TEM, carboxysomes, thylakoids and polyphosphate granules were observed in L. tenuis cells. The
carboxysomes were spatially ordered in a linear fashion in the cells. By IFA, the carboxysomes
were also spatially ordered in a linear fashion in the cells. Carboxysomes are proteinaceousbiochemical compartments of the cyanobacterial CO2-concentrating mechanism. The organelles
catalyze HCO3- dehydration and photosynthetic CO2 fixation. As a result, there is a highpossibility that the granules are the carboxysome.
(2) Development of a new cryogenic sample chamber
A new x-ray cryogenic sample chamber was developed. Temperature stability was improvedby introducing non-contact cooling system with the use of liquid nitrogen and oxygen gas. The
lowest sample chamber temperature reached -170 °C. Alleviation of radiation damage inpolystyrene latex spheres at cryogenic temperatures was verified. The new cryogenic sample
chamber also successfully made experimental observation of cyanobacteria at cryogenic
temperature.
2. Protein folding process
SrcSH3 forms α-helix-rich intermediate in the early stage of folding pathway. Refolding
experiments were done by circular dicroism and X-ray solution sxcattering. Molecular structure of
the folding intermediate was calculated by SAXS_MD program with considering helix-coiltransition theory. Obtained structure well satisfies experimental results of X-ray solution scatteringand circular dichroism.
hNck2 SH3 domain was also studied. This protein takes -structure rich conformation at
native condition. It is known that hNck2 SH3 domain takes equilibrium -helix-rich intermediate
at acidic pH. In our study, it is suggested that the protein takes monomer at acidic pH, and theprotein changes from monomer to mixture of monomer and dimmer with the increase of pH.
From concentration dependence experiment, protein forms monomer at low protein concentrationand forms dimmer at high protein concentration.
-116-

1. 軟 X 線顕微鏡による細胞内機能イメージング①カルボキシソームの X 線顕微イメージング
カルボキシソームは、ルビスコを集積したタンパク質の小構造体で、シアノバクテリ
アや一部の化学合成独立栄養細菌の細胞質に存在する。カルボキシソームは、細胞質に濃縮される重炭酸イオンを CO2 に局所的に変換してルビスコに供給し、炭酸固定反応を助ける働きをする。染色や薄片化していない微小シアノバクテリアの細胞内微細構造を
軟 X 線顕微鏡で、酸素の K 吸収端の前後のエネルギーで観察することにより、カルボキ
シソームと思われる構造体を可視することができた。
②軟 X 線顕微鏡用クライオ試料ステージの開発
液体窒素と酸素ガスを用いることでクライオガスを用いた間接冷却法を開発することで、短時間に試料を試料槽温度で-170℃に冷却することができた。ポリスチレンラテック
ス球を用い冷却による放射線損傷の軽減を確認し、シアノバクテリアのクライオ観察に成功した。
2. X 線溶液散乱による蛋白質のフォールディング過程の追跡
SH3 ドメインのフォールディング過程の追跡を行った。これまで src SH3, PI3K SH3,Fyn SH3 ドメインのような天然構造で β 構造優位な蛋白質においても、フォールディングの初期に非天然な α-helix を形成して巻き戻る事を見出してきた。
今回、src SH3 に関して、フォールディング過程に現れる α-helix に富んだ中間体の構造を明らかにする事が出来た。円偏光二色性、X 線溶液散乱、helix-coil 転移計算、
SAXS_MD 等の手法を組み合わせ、実験結果を満たすフォールディング中間体の構造を求めた。
hNck2 SH3 ドメインに関して新たに研究をスタートした。他の SH3 と同様、フォールデ
ィング過程初期に α-helix を形成して巻き戻る事が期待されたが、リフォールディングの実験からははっきりと分かる α-helix バースト相は確認できなかった。このタンパクは酸性 pH 環境下で構造変化をおこし α-helix に富んだ構造をとる事が知られており、円偏光
二色性の実験にて我々も確認した。又、酸性 pH で単量体だったタンパク分子が、pH 上
昇に伴い単量体と二量体の混合状態になる事、低タンパク濃度では単量体だが、タンパ
ク濃度 1mg/ml 以上の領域では二量体を形成しているであろう事を X 線溶液散乱の結果か
ら見出している。

2. List of Publications
Original Articles
1. K. Takemoto, M. Kimura, K. Usui, T. Ohigashi, H. Fujii, K. Nakanishi, H. Namba and H.
Kihara, Observations of biological specimens at cryo-temperatures with soft X-ray microscopeat the SR center of Ritsumeikan University AIP Conference Proceedings, 1365 (2011)419-422.
2. K. Takemoto, I. Narumi, K. Satoh, T. Ohigashi, H. Namba and H. Kihara, New approach for
x-ray micro-imaging of live cells in carbon window region at subzero temperatures with theuse of antifreeze AIP Conference Proceedings,1365 (2011) 415-418.
3. Kuniko Takemoto, Satoshi Ichise, Takuji Ohigashi, Hidetoshi Namba and Hiroshi Kihara,
X-ray Imaging of Mucilaginous Sheath of Phytoplankton in Lake Biwa by Soft X-ray
Microscope AIP Conference Proceedings, 1365 (2011) 373-376.
4. T. Ohigashi, H. Fujii, K. Usui, H. Namba, H. Mizutani, K. Takemoto, and H. Kihara,
Development of Computer Tomography System for the Soft X-ray Microscope at Ritsumeikan
University AIP Conference Proceedings, 1365 (2011) 124-127.
5. 竹本邦子, 一瀬諭, 大東琢治, 池谷仁里, 難波秀利, 木原裕, 軟Ｘ線顕微鏡を用いた微小植物プランクトンの観察 日本水処理生物学会誌, 47 (2011) 131-135.
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6. Yoshitaka Matsumura, Masaji Shinjo, Nobuyuki Okishio and Hiroshi Kihara, Time-resolved
kinetic refolding of PI3K SH3 in the presence of 45% ethylene glycol at pH 6 Photon Factory
Activity Report, 28:247.
7. Zie-jie Qin, Yoshitaka Matsumura, Masaji Shinjo, Hiroshi Kihara, Structural analysis of
α-helix rich intermediate of bovine β-lactoglobulin on its folding pathway Photon Factory
Activity Report, 28 (2011) 246.
8. Yoshitaka Matsumura, Masaji Shinjo, Anjali Mahajan, Ming-Daw Tsai and Hiroshi Kihara,
Time-resolved SAXS on Ki67 FHA domain refolding at cryo conditions Photon Factory
Activity Report , 28 (2011) 245.
9. Yoshitaka Matsumura, Masaji Shinjo, Nobuyuki Okishio and Hiroshi Kihara, Unfolding of
PI3K SH3 in the presence of 45% ethylene glycol at pH 6 Photon Factroy Activity Report, 28(2011) 248.
10. Yoshitaka Matsumura, Masaji Shinjo, Anjali Mahajan, Ming-Daw Tsai and Hiroshi Kihara,
Time-resolved SAXS on FHA1 domain of Rad53 refolding at cryo conditions Photon Factroy
Activity Report , 28 (2011) 244.
11. Masaji Shinjo, Yasumasa Morimoto, Masaki Kojima, Jinsong Li, Xianju Jin, Yoshitaka
Matsumura, Hiroshi Kihara, Structural analysis of srcSH3 intermediate on its folding pathway
Photon Factory Activity Report, 28 (2011) 242.
12. Kishin Usui, Takuji Ohigashi, Hidetoshi Namba, Kuniko Takemoto, Hiroshi Kihara,
Development of the New Sample Cooling System for the Full-filed Imaging Soft X-ray
Microscope Memoirs of the SR Center, Memoirs of the SR Center, Ritsumeikan University, 13(2011) 207-209.
13. K. Takemoto, S. Ichise, T. Ohigashi, H. Namba, H. Kihara, Soft x-ray imaging of
Leptolyngbya tenis (Phormidium tenue) in Lake Biwa Memoirs of the SR Center, Memoirs of
the SR Center, Ritsumeikan University, 13 (2011) 205-206.
14. Takuji Ohigashi, Hidetoshi Numba, Kuniko Takemoto, Hiroshi Kihara, Improvement of
Illumination Optics of the Full-field Imaging Soft X-ray Microscope, Memoirs of the SR
Center, Ritsumeikan University, 13 (2011) 201-204.
15. Hiroki Fujii, Takuji Ohigashi, Hidetoshi Namba, Kuniko Takemoto, Hiroshi Kihara, Study ofquantitative analytical method for soft X-ray nanotomography at BL-12 Memoirs of the SR
Center, Memoirs of the SR Center, Ritsumeikan University, 13 (2011) 197-200.
Proceedings
1. Masaji Shinjo, Yasumasa Morimoto, Masaki Kojima, Jinsong Li, Xianju Jin, Yoshitaka
Matsumura, Hiroshi Kihara, Structural analysis of α-helix-rich intermediate of src SH3 on its
folding pathway,日本蛋白質科学会, 吹田市, 2011 年 6 月.
2. Yoshitaka Matsumura, Nobuyuki Okishio and Hiroshi Kihara, Multiple folding intermediatesof three SH3 domains (src, Fyn and PI3K) , ,日本蛋白質科学会, 吹田市, 2011 年 6 月.
3. 臼井規真, 大東琢治, 難波秀利, 竹本邦子, 木原裕, 結像型軟 X 線顕微鏡ビームラインの現,立命館大学 SR センター成果報告会, 草津市, 2011 年 6 月.
4. 竹本邦子, 一瀬論, 大東琢治, 難波秀利, 木原裕, 軟Ｘ線顕微鏡による植物プランクト
ン Leptolyngbya tenuis (Phormidium tenue) の顆粒の観察, 立命館大学 SR センター成果
報告会, 草津市, 2011 年 6 月.
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5. 鳴海一成, 佐藤勝也, 大東琢治, 竹本邦子, 難波秀利, 木原裕, 軟 X 線顕微鏡によるDeinococcus radiodutans の観察, 立命館大学 SR センター成果報告会, 草津市, 2011 年 6
月.Yoshitaka Matsumura, Masaji Shinjo, Jinsong Li, Xianju Jin, Kaoru Ichimura and Hiroshi
Kihara, Time-Resolved Study on Folding Landscapes of SH3 Domain Proteins, PF シンポジ
ウム, つくば市, 2011 年 7 月.
6. 新庄正路, Zhi-jie Qin, 松村義隆, Jinsong Li, 木原裕, Structural analysis of α-helix richintermediate of bovine β-lactoglobulin on its folding pathway, PF シンポジウム, つくば市, 2011 年 7 月.
7. Yoshitaka Matsumura, Xianju Jin, Jinsong Li, Masaji Shinjo, and Hiroshi Kihara, Radiation
protection effect of ethylene glycol and glycerol on proteins, PF シンポジウム, つくば市,
2011 年 7 月.
8. 新庄正路, 森本康幹, 小島正樹, Jinsong Li, Xianju Jin, 松村義隆，木原裕, srcSH3 ドメイにおける初期フォールディング中間体の構造解析 , バイオイメージング学会, 千歳市, 2011 年 9 月.
9. 森本康幹, 小島正樹, 新庄正路, 木原裕, srcSH3 ドメインにおける初期フォールディン
グ中間体のイメージング, バイオイメージング学会, 千歳市, 2011 年 9 月.
10. 竹本邦子, 山本章嗣, 大東琢治, 一瀬諭, 難波秀利, 木原裕, 軟Ｘ線顕微鏡による植物
プランクトン Leptolyngbya tenuis (Phormidium tenue) の細胞内顆粒の観察, バイオイメ
ージング学会, 千歳市, 2011 年 9 月.
11. 大東琢治, 臼井規真, 難波秀利, 竹本邦子, 木原裕, 立命館大学 SR センター結像型軟X 線顕微鏡の高度化, X 線結像光学研究会, 仙台市, 2011 年 11 月.
12. 竹本邦子, 山本章嗣, 大東琢治, 一瀬諭, 難波秀利, 木原裕, 軟Ｘ線顕微鏡による植物
プランクトン Phormidium tenue の観察, 日本水処理生物学会, 草津市, 2011 年 11 月.
13. Masaji Shinjo, Yasumasa Morimoto, Masaki Kojima, Jinsong Li, Xianju Jin, Yoshitaka
Matsumura, Hiroshi Kihara, Structural Analysis of alpha-Helix-Rich Intermediate of src SH3
on its Folding Pathway by SAXS_MD, The 5th International Symposium “Molecular Scienceof Fluctuations toward Biological Functions”, Nara, Japan, 2012 年 1 月.
14. Yoshitaka Matsumura, Kaoru Ichimura, Jianxing Song, Hiroshi Kihara, Folding of hNck2 SH3Domain, The 5th International Symposium “Molecular Science of Fluctuations toward
Biological Functions”, Nara, Japan, 2012 年 1 月.
15. 臼井規真, 竹本邦子, 吉村真史, 大東琢治, 木原裕, 難波秀利, 結像型軟 X 線顕微鏡における低ドリフト試料冷却装置の改良と試料観察, 日本放射光学会, 鳥栖市, 2012 年 1月.
16. 竹本邦子, 山本章嗣, 大東琢治, 一瀬諭, 難波秀利, 木原裕, 軟Ｘ線顕微鏡による植物
プランクトン Leptolyngbya tenuis の細胞内微細構造の観察と同定, 日本放射光学会,
鳥栖市, 2012 年 1 月.

-119-Annual Report 研究成果報告書

Title of Project

Molecular imaging of pathophysiology of kidney and neurodevelopmental
disorders
腎不全とてんかん・発達遅滞に関わる疾患遺伝子の病態分子イメージングの開発

P.I.

Hiroyasu Tsukaguchi, 塚口裕康

Affiliation

Medical Informatics, 医療情報部

E-mailtsukaguh@takii.kmu.ac.jp

Collaborators
Keywords

disease-specific iPS, molecular imaging, neuron, PET, podocyte

1. SUMMARY OF RESEARCH RESULTS[Background] A neuron and glomerular podocyte share a common biological feature of terminallydifferentiated cells that exert their function through formation of specialized cell-cell junctions(i.e., synapse and slit diaphragm). Galloway-Mowat syndrome (GMS, MIM 251300) is a rarecongenial disorder that affects these two cellular lineages simultaneously.
Clinically, it is recognized by co-occurrence of malformation in cerebral cortex and renal
glomeruli. The GMS patients typically suffer from nephrotic syndrome with progression to end
stage renal disease and from psychomotor retardation with epilepsy. The pathogenesis might beascribed to defective cellular functions shared by neuronal and renal mesenchymal lineages, i.e.,
migration, proliferation. However, the disease genes have yet to be identified.
[Purpose] The aim of this study is, (1) to identify as-yet-undefined disease genes of GMS, (2) togenerate an experimental disease model of mice and iPS cells, and (3) to create a functional
imaging system that visualize the pathogenic events in vivo.
[Results & Methods](1) Genetic analysis: We searched the disease genes of 10 Asian GMS families by combination of
cytogenetic microarray (300K CytoSNP-12, Illumina) and next generation sequencing of wholeexomes (4-5Gb, 75bp-paired ends with x30-50 depth). After filtering against SNP databases (1000genomes), there were several potential candidate genes. We are now on the process of verifying
their significance in terms of segregation and tissue localization.
(2) Disease-specific iPS cells: The iPS cells were successfully generated from the patient’s skinfibroblasts through transfection of defined Yamanaka four factors by Sendai-virus vectors (Fusaki
N, 2009). The cells are now being tested to induce the differentiation states towards neuronal andrenal progenitors.
[Conclusions]
Discovery of the GMS genes will lead to a better understanding of pathophysiology regarding
neuron and podocyte development, thereby enabling the earlier diagnosis and more targeted
therapy onto this rare syndromic disorder. Identification of GMS genes, the first step of thisproject, will allow the generation of probes (fluorescence or radionucleotides) that could monitor aspatiotemporal expression of the causative molecules. We will then proceed with developing
non-invasive, real-time imaging system that envisions the pathogenic events resulting from the
GMS genes, at both cellular (iPS) and rodent models (transgenic mice). This new technology will
hopefully improve the treatment for more common diseases, i.e., chronic kidney disease (CKD)
and epilepsy.
中枢（精神発達遅滞、てんかん）と腎糸球体（ネフローゼ、腎不全）障害を主徴候とす-120-

る原因不明の稀少難病 Galloway-Mowat 症候群の責任遺伝子を同定し、生体内で原因分子
および疾患にともなう病態変化を可視化する画像診断技術の基盤を構築する。
[背景・準備状況] 主研究者らは、Galloway-Mowat 症候群の全国調査と検体収集を行い、疾患遺伝子の探索を行っている（平成 22 年厚労省難治性疾患克服研究事業）。
[目的] Galloway-Mowat 症候群は、ニューロンと糸球体上皮ポドサイトに共通する分化・発育因子の異常に起因する脳・糸球体異形成であるが、原因分子は不明である。次世代シークエンサを用いたエクソーム解析で責任遺伝子を同定する。疾患分子の情報を基に、腎疾患、てんかん・発達障害の病態診断・治療に役立つ新しい生体分子イメージング技術（PET等）を開発する。
[成果]
1) 患者調査・疾患遺伝子探索 患者情報を収集するとともに 10 症例の SNP マイクロアレ
イと次世代シークエンサを用いた全エクソーム解析を行い、疾患遺伝子を解析中である（塚口、金子）。
2) 疾患モデル幹細胞の作成 Galloway-Mowat 症候群患者で、iPS 細胞を樹立した（江良、仲里）。現在、神経細胞や腎前駆細胞（中間中胚葉）への誘導を試み、病態モデルとしての評価を行っている。
[意義]1) 学術的独創性・新規性 ニューロンとポドサイトの分化・発育、組織構築に必須の分子の実体は不明であり、本プロジェクトにより新たな生命現象の解明と、研究領域の創生が期待できる。
2) 医学的貢献 脳神経や腎糸球体の発達経過と病態を、非侵襲的、経時的に可視化できる
病態分子イメージング技術は、早期診断、治療効果判定、創薬開発に役立ち、臨床の現場に貢献できる。
3) 社会的貢献 成果は難治性てんかん、発達障害、進行性腎障害（有病率 1%）の治療開発に役立ち、広く社会に貢献する。

2. List of Publications
Original Articles
1. 塚口裕康 腎臓病学「ゲノムからみた腎臓病学」

細胞 5 月号 2012

2. 塚口裕康 Galloway-Mowat 症候群(脳・腎糸球体異形成) 別冊 日本臨床 腎臓症候群(上): page 411-419
Proceedings
1. 塚口裕康、仲里仁史、服部元史、伊藤秀一、小崎里華, 飯島一誠 Galloway-Mowat 症候群(脳腎糸球体異形成) の全エクソーム解析、第 56 回日本人類遺伝学会 千葉幕張
2011 年 11 月 13 日
2. 塚口裕康、遺伝性・症候性ネフローゼ症候群の遺伝子解析 第 115 回小児科学会学術集会、シンポジウム 福岡国際会議場 2012 年 4 月 21 日
3. 塚口裕康、分子病態理解に基づく難治性ネフローゼ克服へのアプローチ、第 47 回小児腎臓病学会学術集会、教育講演 東京都市センターホテル 2012 年 6 月 29 日

-121-

-122-

Research Activities 2012
平成 24 年度研究成果報告

Division of Neuroscience

神経部門

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Annual Report 研究成果報告書
Title of Project

In situ imaging of neural cells and their function
神経細胞機能の in situ イメージング

P.I.

Hisao Yamada, 山田久夫

E-mailyamada@hirakata.kmu.ac.jp

Affiliation

Regenerative Neural Medicine, 神経機能再生医学（解剖学第 1）講座

Collaborators

若林毅俊、森徹自、高森康晴、平原幸恵、小池太郎

Keywords

BrdU, IdU, subventricular zone, neurogenesis, mouse, cell cycle

1. SUMMARY OF RESEARCH RESULTS
Adult neurogenesis in the subgranular zone of the hippocampus (SGZ) is enhanced by excess aswell as mild neuronal excitation, such as chemoconvulsant-induced brief seizures. Because moststudies of neurogenesis after seizures have focused on the SGZ, the threshold of neuronal
excitation required to enhance neurogenesis in the subventricular zone (SVZ) is not clear.
Therefore, we examined the responses of SVZ precursors to brief generalized clonic seizures
induced by a single administration of the chemoconvulsant pentylenetetrazole (PTZ). Cell cycle
progression of precursors was analysed by systemic administration of thymidine analogues. We
found that brief seizures immediately resulted in cell cycle retardation in the SVZ.
However, the same effect was not seen in the SGZ. This initial cell cycle retardation in the SVZ
was followed by enhanced cell cycle re-entry after the first round of mitosis, leading to precursor
pool expansion, but the cell cycle retardation and expansion of the precursor pool were transient.
Cell cycle progression in the PTZ-treated group returned to normal after one cell cycle. The
numbers of precursors in the SVZ and new neurons in the olfactory bulb, which are descendantsof SVZ precursors, were not significantly different from those in control mice more than 2 days
after seizures. Because similar effects were observed following electroconvulsive seizures, these
responses are likely to be general effects of brief seizures. These results suggest that neurogenesis
in the SVZ is more tightly regulated and requires stronger stimuli to be modified than that in the
SGZ.
われわれは、神経組織での細胞新生と分化を追究する一連の研究をおこなっているが、今回は以下の成果を紹介する。
成個体のニューロン新生は、例えば痙攣誘発性薬剤による短期発作といったニューロン
興奮で海馬歯状回顆粒層(SGZ)における新生が増強されるので、この方面の研究は SGZ に集中しておこなわれてきた。従って、側脳室周囲のニューロン新生領域(periventriculargerminal zone＝PVGZ、いわゆる脳室下帯＝SVZ)におけるニューロン過剰興奮とニューロ
ン新生の関係は明らかになっていない。
われわれは、痙攣誘発化学物質ペンチレンテトラゾール(PTZ)の単回投与によって誘発される全身性間代発作を起こさせたマウスを用い、SVZ 神経前駆細胞の細胞周期進行を、チミジン類似体 BrdU/IdU を系統的に投与することにより検索した。
発作をおこさせると、直ちに SVZ 神経前駆細胞の細胞周期進行が遅延したが、SGZ では遅延はおこらなかった。この最初の細胞周期遅延の後に、神経前駆細胞の細胞周期への再入が促進されることにより前駆細胞プールが拡大するが、これらの現象は一過性であった。またこの細胞周期の進行遅延は１周期で終了し、通常のサイクルに戻った。SVZ における前駆細胞数と、この前駆細胞が移動・分化した嗅球の新生ニューロン数は、痙攣発
作後には、コントロールマウスとの間で有意差がなかった。電気刺激による痙攣発作でも同様の現象がみられたので、得られた現象は痙攣発作の際の細胞周期にとって一般的な現象と考えられる。
このような結果は、SVZ のニューロン新生は厳密に調整されていて、その修正には SGZ-124-

の場合よりもより強い刺激を必要とすることを示唆している。
優れた成果があがった点
成個体脳内での神経前駆細胞の細胞周期に関する新たな知見が得られ、論文として発表
した。
問題点
特になし

2. List of Publications
Original Articles
1. Cui, Y., Li, Q-H., Yamada, H., Watanabe, Y. and Kataoka, Y. Chronic Degeneration of
Dorsal Raphe Serotonergic Neurons Modulates Cortical Spreading Depression: A Possible
Pathophysiology of Migraine. J. Neurosci. Res. レフェリー有, 91, 2013, 737–744.

2. Hirahara, Y., Matsuda, K.I., Liu, Y.F., Yamada, H., Kawata, M. and Boggs, J.M.
17β-Estradiol and 17α-Estradiol Induce Rapid Changes in Cytoskeletal Organization in
Cultured Oligodendrocytes. Neuroscience, レフェリー有, 235, 2013, 187-199.
3. Hirahara, Y., Matsuda, K.I., Yamada, H., Saitou, A., Morisaki, S., Takanami, K., Boggs, J.M.
and Kawata, M. G protein-coupled receptor 30 contributes to improved remyelination after
cuprizone-induced demyelination. Glia, レフェリー有, 61, 2013, 420-431.
4. Kimura, T., Jiang, S., Nishizawa, M., Yoshigai, E., Hashimoto, I., Nishikawa, M., Okumura, T.
and Yamada, H. Stabilization of human interferon-α1 mRNA by its antisense RNA.
Cell Mol. Life Sci. レフェリー有, in press, 2012.
5. Mori, T., Wakabayashi, T., Hirahara, Y., Takamori, Y., Koike, T., Kurokawa, K. and Yamada,H. Differential responses of endogenous adult mouse neural precursors to excess neuronal
excitation. Eur. J. Neurosci. レフェリー有, 36, 2012, 3184-3193.
6. Wakabayashi, T., Kosaka, J., Mori, T. and Yamada, H.
Prolonged Expression of Puma in Cholinergic Amacrine Cells During the Development of Rat
Retina. J. Histochem. Cytochem. レフェリー有, 60, 2012, 777-788.
Proceedings
1. Hirahara, Y., Wakabayashi, T., Mori, T., Matsuda, K., Koike, T., Takamori, Y., Kawata, M. and
Yamada, H. Expression and function of the G protein-coupled receptor 30 in oligodendrocyte.
14th International Congress of Histochemistry and Cytochemistry, Kyoto, 2012.8.

2. Koike, T., Wakabayashi, T., Mori, T., Hirahara, Y., Takamori, Y. and Yamada, H.
Histochemical demonstration of Sox2 and cell-markers in sensory nervous system.
14th International Congress of Histochemistry and Cytochemistry, Kyoto, 2012.8
3. Mori, T., Wakabayashi, T., Hirahara, Y., Takamori, Y., Koike, T., Kurokawa, K. and Yamada,H. Cell cycle analysis of neural precursors in the adult mouse subventricular zone.
14th International Congress of Histochemistry and Cytochemistry, Kyoto, 2012.8.

-125-Annual Report 研究成果報告書
Title of Project

Striatal neurons highly expressing GAD1 mRNA are populated in the lateral
striatum
マウス線条体における calbindin-poor 領域の空間配置と GABA ニューロン

P.I.

Tetsuo Sugimoto, 杉本哲夫

Affiliation

Anatomy and Brain Science, 脳構築学（解剖学第 2）講座

Collaborators

Trifonov Stefan、山下雄司、丸山正人、加瀬政彦

Keywords

GPR155, splicing variant, GABA neuron, lateral striatum

E-mailsugimoto@hirakata.kmu.ac.jp

1. SUMMARY OF RESEARCH RESULTS
Integral membrane protein GPR155 is a novel member of the group of proteins containing
DEP domain. It is expressed in the mouse striatum, hippocampus and cerebral cortex, andchanges in its expression have been related to autism-spectrum disorders and Huntington's
disease. Molecular mechanisms associated with the localization and function of GPR155 in the
striatum have yet to be elucidated. We have shown that GPR155 in the mouse has splicing
variants 1 to 5, in which variant 1 is dominant in the brain. We here demonstrate that the neurons
highly expressing the variant 1 are dominantly accumulated in the V-shaped stripe structure inmouse lateral striatum delineated by its low expression of Calbindin D28K. In this study, we have
prepared rabbit anti-peptide antibodies against mouse GPR155 and examined its distribution on
immunostained tissue sections of mouse striatum. To visualize the V-shaped stripe structure, wehave immunostained the tissue sections with Calbindin D28K antibodies. GPR155-heavily
immunopositive neurons were found in the lateral striatum largely conforming to the regions of
the V-shaped stripe structure visualized by the reduced Calbindin D28K immunoreactivity.
GPR155-moderately immunopositive neurons were distributed in the surrounding areas including
the central and medial striatal regions. The majority of GPR155-immunopositive cells appeared
as medium spiny projection neurons. GPR155-immunopositive fibers were accumulated inregions of external and internal segments of the globus pallidus and the substantia nigra pars
reticulata. Taken together with the results from in situ hybridization, GPR155-expressing neurons
might be projection neurons highly expressing GAD1 in the lateral striatum. These projection
neurons may contribute to basal ganglia circuits regulating motor execution.
マウス・ラットの線条体において in situ hybridization 法で GABA ニューロンの GAD 発現を検出したところ、特に線条体外側部において中型有棘ニューロン集団が GAD1mRNA を高度に発現していることを見つけ出した。一方、GAD2 に対する in situ
hybridization を試みたところ、GAD1 で示されたような特異的集積像は得られなかった。
また、hybridization には、GAD1、GAD2 の coding region に対する riboprobe に加えて5'-noncoding region および 3'-noncoding region に対する riboprobe を用い判定に慎重を期したが、いずれの結果においても線条体外側部の中型有棘ニューロン集団が GAD1 mRNA
を高度に発現していた。これと一致して、免疫染色により GAD1 陽性エレメントを検出したところ、in situ hybridization の結果と一致して線条体外側部に特に増強していることを見つけ出した。線条体外側部（被殻に相当する領域）は機能的には運動遂行の制御を担当する領域である。この領域は幾つかの点において他の線条体領域とは異なる特殊な線条体領域とみなすことができた。第一に、カンナビノイド 1 型受容体を高度に発現するニューロンが集中した。第二に、GPR155 タンパクを高度に発現するニューロンが局在した。第三に、大脳皮質との連絡を見た場合、GAD1 mRNA を高度に発現している領域は、口腔、顔面から前肢に対応する一次運動皮質の入力を比較的強く受けることがわかった。第四に、線条体外側部は patch-matrix 構造を呈しない領域に属することが判明し
た。
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優れた成果があがった点
GPR155 マーカー発現などの新しいイメージング手法を用いて、線条体外側部ニューロンの特徴を明らかにした点。
問題点
特になし。

2. List of Publications
Original Articles
1. Maruyama, M., Yamashita, Y., Kase, M., Trifonov, S. and Sugimoto, T.
Lineage-specific purification of neural stem/progenitor cells from differentiated mouse
induced pluripotent stem cells.
Stem Cells Trans. Med. レフェリー有, in press, 2013.

2. Hamada, S., Trifonov, S., Houtani, T., Kase, M., Yamashita, Y., Baba, K., Shimizu, J.,
Maruyama, M., Tomoda, K., Yamashita, T. and Sugimoto, T.
Cholinergic fibers and muscarinic receptors in the central auditory system.
J. Kansai Med. Univ. レフェリー有, 63, 2012, 7-9.
3. Trifonov, S., Houtani, T., Kase, M., Toida, K., Maruyama, M., Yamashita, Y., Shimizu, J. and
Sugimoto, T.
Lateral regions of the rodent striatum reveal elevated glutamate decarboxylase 1 mRNA
expression in medium-sized projection neurons.
Eur. J. Neurosci. レフェリー有, 35, 2012, 711-722.
4. Horie, A., Fujiwara, H., Sato, Y., Suginami, K., Matsumoto, H., Maruyama, M., Konishi, I.
and Hattori, A.
Laeverin/aminopeptidase Q induces trophoblast invasion during human early placentation.
Hum. Reprod. レフェリー有, 27, 2012, 1267-1276.
Proceedings
1. 山下雄司, Stefan Trifonov, 丸山正人, 加瀬政彦, 杉本哲夫.
GPR 抗体を用いたマウス線条体外側部 V 字型縞構造の解析．
第 118 回日本解剖学会総会・全国学術集会, 高松, 2013.3

2. 丸山正人, 山下雄司, Stefan Trifonov, 加瀬政彦, 杉本哲夫.
神経幹細胞特異的に発現させた薬剤耐性遺伝子によるマウス iPS 細胞由来神経幹細胞の純化．
第 133 年会日本薬学会, 横浜, 2013.3
3. 山下雄司, Stefan Trifonov, 丸山正人, 加瀬政彦, 杉本哲夫.
マウス線条体 calbindin-poor 領域：中型ニューロンの選択的配置．
第 35 回日本神経科学大会, 名古屋, 2012.9-127-

Annual Report 研究成果報告書
Title of Project

Imaging of disease-related molecules and cells
病因細胞および分子のイメージング

P.I.

Hiroko Matsuda, 松田博子

E-mailmatsudah@hirakata.kmu.ac.jp

Affiliation

Cellular and Molecular Physiology, 細胞分子生理学（生理学第 1）講座

Collaborator

松田博子、岡田誠剛、武藤恵、林三樹夫

Keywords

K+ channel, pancreatic duct, electrophysiology, GH

1. SUMMARY OF RESEARCH RESULTS
Expression of KCa3.1 channel in the pancreatic duct cells
Potassium channels play a vital role in maintaining the membrane potential and the driving
force for anion secretion in epithelia. In pancreatic ducts, however, identity of K+ channels
has not been extensively investigated. Therefore, the aim of our study was to functionallyidentify K+ channels that contribute to secretion in pancreatic ducts. The molecular basis of
K+ channels is revealed using RT-PCR analysis and immunostaining. We measured
equivalent short-circuit current (Isc), membrane potential, and single-channel currents using
electrophysiological techniques. Pancreatic duct cells expressed KCNN4 coding for anintermediate-conductance Ca2+-activated K+ channel (KCa3.1). KCa3.1 protein localized inluminalandbasolateral
membranesofductcells.
DC-EBIO(5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazole-2-one), an activator of KCa3.1 channel,
increased Isc in Capan-1 monolayer. The effect was consistent with Cl− secretion in epithelia.
Clotrimazole, a blocker of KCa3.1 channel, inhibited Isc. KCa3.1 channel blockers
depolarized the membrane potential of cells in microperfused ducts dissected from rodent
pancreas. Cell-attached patch clamp single-channel recordings revealed KCa3.1 channels withan average conductance of 80 pS in freshly isolated duct cells. In conclusion, these results
indicated that the KCa3.1 channels may, at least in part, be involved in setting the resting
membrane potential. Furthermore, the KCa3.1 channels are involved in anion transport instimulated pancreatic ducts.
Lentiviral and Moloney retroviral expression of green fluorescent protein in somatotrophs
in vivo
Previous studies have shown that the locus control region (LCR) and the promoter of thegrowth hormone (GH) gene can control the expression of GH. Therefore, lenti- and retro-viral
vectors with these elements might be useful to monitor the activation of the GH gene and thedevelopment of newborn somatotrophs. To test this, we first constructed a lentiviral vector,
which expresses green fluorescent protein (GFP) under the control of these elements, andinjected them into rat pituitaries in situ and in vivo. The lentiviral vector expressed GFP
specifically in the anterior lobe, and nearly all GFP-positive cells were anti-GH
immunoreactive. The GFP expression was upregulated by the administration of growth
hormone releasing hormone and an IGF-1 receptor blocker. Furthermore, the social isolationstress, which was shown to decrease the GH secretion, decreased the GFP expression. Second,
we injected the retroviral vector into neonatal rat pituitaries in vivo. At 30 days postinjection(DPI), almost all GFP-positive cells were anti-GH positive and anti-prolactin negative as thelentiviral expression. However, GFP was transiently expressed by developing lactotrophs at 8
and 16 DPI, suggesting that our vector lacks an element(s) which suppresses the expression.
Meanwhile, the retrovirally labeled cells tended to cluster with the cells of same type. An analysis
of cell numbers in each cluster revealed some features of cell proliferation. These viral vectors are-128-shown to be useful tools to monitor the activation of the GH gene and the development ofsomatotrophs.
High-through-put assay for inwardly rectifying K+ channel
Based on a finding that overexpression of inwardly rectifying K+ channel (Kir2.1) decreases the
coexpression of GFP, we established a high-through-put assay for Kir2.1. We have screened thesamples from venom of scorpions and spiders and botanical extracts, from which many ion
channel modifiers have been found. We found a blocking compound and confirmed current
blockade.
膵導管細胞に発現する Ca2+依存性 K+チャネル
K+チャネルは負の膜電位を維持することで、上皮細胞における陰イオン分泌に駆動力を与える。重炭酸イオンを分泌する膵臓導管上皮細胞での K+チャネルの分子基盤については不明の点が多い。本研究で、膵臓導管細胞に機能発現する K+チャネルを明らかにした。
まず RT-PCR 法を用いて、KCNN4 分子の発現を認めた。次に免疫組織化学法を用いて、KCNN4 がコードする KCa3.1 蛋白が管腔側膜および血管側膜に分布することを観察した。
さらに導管細胞の単層培養を用いた短絡電流測定において、KCa3.1 チャネル機能に依存す
る上皮膜イオン輸送を認めた。また、KCa3.1 チャネルの遮断薬は新鮮分離導管細胞の静止
膜電位を脱分極させた。それに加えて導管細胞の血管側膜に、内向き整流性 K+コンダク
タンスを認めた。これらの結果は、KCa3.1 チャネルが導管細胞の膜電位を維持することで膵液分泌に寄与する可能性を示唆した。
レンチ及びレトロウイルスを用いた GFP のソマトトロフでの発現
成長ホルモンの分泌は、発達、加齢、ホルモンの影響を受けるだけでなく、身体的、精神的ストレスによっても変化する。我々は、成長ホルモン遺伝子の locus control region とプロモーターの下流に緑色蛍光タンパク(GFP)遺伝子を挿入したレンチ及びレトロウイル
スベクターを作製し、in vivo で GFP 発現を検討した。同レンチウイルスをラット下垂体に注入すると、GFP は前葉の抗 GH 免疫陽性細胞に発現し、GHRH 及び IGF 受容体遮断薬によって発現が増加した。幼弱ラットの隔離飼育ストレスは、GFP 発現を低下させた。
次に、同レトロウイルスで、新生細胞で検討し、GFP は一過性にラクトトロフに発現すること、並びに、同じ幹細胞に由来する細胞はクラスターを形成し、クラスター内の細胞は、分裂前にソマトトロフに分化するか否かが決定していることを明らかにした。
内向き整流性 K+チャネルの遮断・開口薬の天然物からの探索
我々は、細胞に過剰に発現させた内向き整流性 K+チャネル(Kir2.1)電流が細胞毒性をきたし、GFP の共発現を減少させることを利用して、同チャネル遮断・開口薬のハイスル
ープット・アッセイを確立し、サソリ、クモ毒、および、植物サンプルからのスクリーニ
ングを行い、遮断活性のある化合物を見出した。
優れた成果があがった点特記するほどの優れた成果は得られなかった。
問題点
特記するほどの問題点はない。
2. List of Publications
Original Articles
1. Hayashi, M., Wang ,J., Hede, S.E. and Novak, I.
An intermediate-conductance Ca2+-activated K+ channel is important for secretion inpancreatic duct cells.
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American Journal of Physiology. Cell Physiol. レフェリー有, 303, 2012, C151−C159.

2. Okada, M., Matsuda, H. and Okimura, Y.
Lentiviral and Moloney retroviral expression of green fluorescent protein in somatotrophs invivo.
PLoS One, レフェリー有, 8, 2013, e54437(1-11).
Proceedings
1. Taketo, M., Matsuda, H.
Changes of spontaneous GABAergic currents after increasing pre- and post-synaptic activities
in rat hippocampus.
第 90 回日本生理学会大会, 東京, 2013.3.

2. Hayashi, M., Wang, J., Hede, S.E., Novak, I.
KCa3.1 channel is important for secretion in pancreatic duct cells.
第 90 回日本生理学会大会, 東京, 2013.3.
3. Inagaki, A., Hayashi, M., Matsuda, H.
Adenosine receptors regulate Cl− channels in pancreatic duct cells.
第 90 回日本生理学会大会, 東京, 2013.3.
4. Hayashi, M., Wang, J., Novak, I., Matsuda, H.
膵液分泌に寄与するカリウムイオンチャネルの同定
2012 年度生理学研究所研究会, 岡崎, 2012.11.
5. Hayashi, M., Wang, J., Novak, I., Matsuda, H.
膵臓導管細胞に機能発現するカリウムイオンチャネル
第 105 回近畿生理学談話会, 守口, 2012.9.
6. Novak, I., Haanes, K.A., Wang, J., Hayashi, M. and Nielsen, L.S.
Purinergic signalling amplifies exocrine pancreatic functions.
International Symposium on Purinergic Signalling in New Strategy of Drug Discovery,
Fukuoka, 2012, June.
7. Novak, I., Wang, J. and Hayashi, M.
Purinergic signalling coordinates function of Cl− and K+ channels in pancreatic secretion.
International Symposium on Purinergic Signalling in New Strategy of Drug Discovery,
Fukuoka, 2012, May.
8. Okada, M., Kano, M. and Matsuda, H.
Current-dependent degradation of the inwardly rectifying potassium channel, Kir2.1.
Gordon Research Conference, Cell Biology of the Neuron, Waterville Valley, USA, 2012,
June.

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Annual Report 研究成果報告書
Title of Project

The role of serotonin and dopamine for cognitive processes in the primate.
霊長類におけるセロトニン・ドパミンによる認知機能の制御

P.I.

Kae Nakamura, 中村加枝

E-mailnakamkae@hirakata.kmu.ac.jp

Affiliation

Cognitive Neuroscience, 高次認知脳科学（生理学第 2）講座

Collaborators

磯田昌岐、上田康雅、則武厚、時田賢一

Keywords

dopamine, serotonin, monkey, reward, doral raphe N., hypothalamus

1. SUMMARY OF RESEARCH RESULTS
1. To understand the neuronal mechanisms of decision making based on rewards and punishments,
we recorded single neuronal activity of neurons in the dorsal raphe nucleus (DRN), the major
source of serotonin, and the lateral hypothalamic area (LHA), one of the main source of dopamine
neuronal activity which encodes reward predication error, while monkeys performed (1) Pavlovian
conditioning task and (2) Choice task.
In the DRN, we found that many neurons showed sustained modulation in activity which
encoded reward context; whether majority of trials were rewarded or punished. The finding
indicates that DRN neurons keep track of ongoing value of events.
The LHA has been implicated in encoding positive value. However, we found some neurons also
responded to punishments. There were also neurons which showed stronger activity for uncertain
situation. Thus LHA neurons exhibited much more variety of reward information than previously
thought.
2. Although much learning occurs through direct experience of errors, humans and other social
animals can learn from the errors of other individuals. Using two paired monkeys that monitoredeach other’s action for their own action planning, we identified a group of neurons in the medial
frontal part of the cerebral cortex that exhibited a selective activity increase upon seeing another’s
errors.
1. 報酬とそれを得るためのコスト・嫌悪刺激の情報を計算して行動を選択する神経メカ
ニズムを明らかにするために、我々は、カニクイザル計 3 頭において、パブロフ型の条件付け課題と、２者択一課題を訓練し、セロトニン細胞の存在する背側縫線核(DRN)、ドパミン系に強い投射がある視床下部外側部(LHA)から単一神経細胞外記録を行った。それぞれの領域では異なる形で報酬や嫌悪刺激情報が表現されていることがあきらかになった。
DRN：パブロフ型の条件付け課題において、持続的な神経の発火がみられるが、報酬が得られる試行が多いコンテキスト（試行の継続）と罰が与えられる試行が多いコンテキス
トによって発火率が大きく異なる神経細胞が多く見られた。そのことから DRN は現在行っている行動の価値を持続的にモニターすると考えられる。
LHA：従来は正の報酬に応答すると考えられていたが、正のみならず負の報酬に応答するものがあり、さらに報酬確率をコードしている細胞群も見つかった。さらに、報酬が与えられるかどうか不確定な場合に発火が強くなる細胞も見られた。このように LHA は従来考えられていたよりはるかに多様な情報処理が行われていることが明らかになった。
2. さらに、セロトニンは個体間の社会性に影響があるという仮説がある。社会性人間を含む社会的動物は、己の過ちのみならず、他人の過ちからも多くを学ぶことが可能である。
このような「他者から学ぶ」能力の神経機構を明らかにするため、2 頭のサルが互いに相手の行動を読み取り、それをもとに自己の最適行動を導いているときに、大脳皮質の神経細胞の活動を記録・解析した。そして、他者の行動の過ちを検出する神経細胞が、前頭葉の内側領域に存在することを突き止めた。
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優れた成果があがった点
報酬に関しての研究は多いが、罰の情報さらには社会的な相互作用に関しての情報処理の研究は非常に遅れている。本研究で、脳の複数の領域でこのような情報処理過程が測定された。
今後は、薬理行動学的な手法も取り入れ、物質的な基盤も明らかにしていきたい。
問題点
特にない。

2. List of Publications
Original Articles
1. Nakamura, K., Santos, G., Matsuzaki, R. and Nakahara, H.
Differential reward coding in the subdivisions of the primate caudate during an oculomotor
task.
J. Neurosci. レフェリー有, 32, 2012, 1518-1512
2. Isoda, M.
Recent advances in social neuroscience research using macaques.
Brain and Nerve. レフェリー有, in press, 2013.
3. Isoda, M.
Using saccades as a probe of higher brain functions.
Neuro Ophthalmol. Jpn. レフェリー有, in press, 2013.
4. McCairn, K.W., Iriki, A. and Isoda, M.
Deep brain stimulation reduces tic-related neural activity via temporal locking with stimulus
pulses.
J. Neurosci. レフェリー有, 33, 2013, 6581-6593.
5. McCairn, K.W., Iriki, A., Isoda, M.
Global dysrhythmia of cerebro-basal ganglia-cerebellar networks underlies motor tics
following striatal disinhibition.
J. Neurosci. レフェリー有, 33, 2013, 697-708.
6. Yoshida, K., Saito, N., Iriki, A., Isoda, M.
Social error monitoring in macaque frontal cortex.
Nat. Neurosci. レフェリー有, 15, 2012, 1307-1312.
7. Yamada, H., Inokawa, H., Matsumoto, N., Ueda, Y., Enomoto, K. and Kimura, M.
Coding of the long-term value of multiple future rewards in the primate striatum.
J. Neurophysiol. レフェリー有, 109, 2013, 1140-1151.
Books
1. McCairn, K.W., Imamura, Y., Isoda, M.
Oxford University Press, Animal models of Tics :Tourette Syndrome (eds.by Martino D&Leckman JF), 2013, 672.
Proceedings
1. Nakamura, K.
Differential reward coding in the dorsolateral and ventromedial part of the primate caudate.
The 42nd Annual Meeting of Society for neuroscience meeting, New Orleans, USA, 2012.10.
2. Noritake, A. and Nakamura, K.
Coding of reward uncertainty in the primate lateral hypothalamic neurons.
The 42nd Annual Meeting of Society for neuroscience meeting, New Orleans, USA, 2012.10.
3. Noritake, A. and Nakamura, K.
Uncertainty coding in the primate lateral hypothalamus area.
The 35th annual meeting, the Japan Neuroscience Society, 名古屋, 2012.9.
4. Hayashi, K., Noritake, A. and Nakamura, K.
Action control and value coding in the primate dorsal raphe nucleus.
The 35th annual meeting, the Japan Neuroscience Society, 名古屋, 2012.9.
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5. Isoda, M.
Monitoring other’s actions in the medial prefrontal cortex of the macaque.
第 3 回前頭前野国際シンポジウム（招待講演）, 京都, 2012.11.
6. 磯田昌岐.
眼球運動を手がかりとして認知機能の脳内機構を探る.
第 50 回日本神経眼科学会総会（招待講演）, 京都, 2012.11.
7. Yoshida, K., Saito, N., Iriki, A. and Isoda, M.
Atypical agent-related activities in the medial frontal cortex of a monkey with impaired social
monitoring.
The 42nd Annual Meeting of Society for neuroscience meeting, New Orleans, USA, 2012.10.
8. McCairn, KW., Iriki, A., Isoda, M.
The cerebellum contributes to basal ganglia mediated motor tics.
第 35 回日本神経科学学会大会, 名古屋, 2012.9.
9. Isoda, M.
Probing the neural correlates of self and others' actions in monkey medial frontal cortex.
Mirror neurons: 20 years after the discovery（招待講演）, 京都, 2012.9.
10. Ueda,Y., Okazaki,S.R., Yamanaka, K., Samejima, K., Enomoto, K., Inokawa, H., Matsumoto,N., Kano, M. and Kimura, M.
Cannabionid(CB1) receptors selectively modulate value of action encoded in the striatum.
The 35th annual meeting, the Japan Neuroscience Society, 名古屋, 2012.9.
11. Ueda, Y., Okazaki, S.R., Yamanaka, K., Enomoto, K., Kano, M. and Kimura, M.
Presentation Session Wed, Impairment of reward-based adaptive choice of actions by local
injection of cannabinoid CB1 receptor antagonist into the striatum of behaving monkeys.
The 42nd Annual Meeting of Society for neuroscience meeting, New Orleans, USA, 2012.10
12. Noritake, A. and Nakamura, K.
Neurons in the primate lateralhypothalamic area encode reward probability, uncertainty,
andpredicted values.
University of Chicago Organismal Biology & Anatomy Seminar Series, Chicago, USA,2012.10.

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Annual Report 研究成果報告書
Title of Project

Elucidation of pathogenesis and development of diagnosis and treatment of
intractable neural diseases
難治性神経疾患の病態解明と診断治療法の開発

P.I.

Seiji Ito, 伊藤誠二

E-mailito@hirakata.kmu.ac.jp

Affiliation

Molecular and Functional Biology, 分子生体機能学（医化学）講座

Collaborators

松村伸治、矢尾育子、下條正仁、片野泰代、西田和彦

Keywords

ACh, imaging mass spectrometry, two-photon microscopy, spinal cord

1. SUMMARY OF RESEARCH RESULTS
1.

Molecular Imaging
1) Neurodegenrative diseases such as Alzheimer’s disease and Parkinson’s disease are causedby neuronal death, loss of function, in a given region. So far, pathological alterations in tissue
samples of postmortum brain and model animals were histochemically examined. Metabolite
distribution imaging via imaging mass spectrometry (IMS) is an increasingly utilized tool in thefield of neurochemistry.We succeeded in measurement of acetylcholine (ACh), a neurotransmitter,by IMS. Its distribution in the brain was well correlated with that of acetylcholinesterase. We
also identified mochi as obstracting substance in formalin-fixed paraffin-embeded samples
prepared from a paitient suffered with intestinal obstraction. At present, 6 departments belonging
to Molecular Imaging Center of Diseases use IMS and have obtained good results gradually.
2) Noxious and non-noxious stimuli are conveyed by prmary afferent fibers which form synapse
with spinal neurons, processed in network of dorsal horn of the spinal cord, transmitted to thecortex, and recognized as pain and touch, respectively. To clarify how sensory reception from theperipheral is processed in the spinal cord, we have analyzed spinal responses functionally andmorphologically using two-photon microscopy and fluorescent protein-expressing mice. Whilein vivo patch clamp can analyze cellular response in only one spinal neuron, two-photon
microscopy can analyze it in many cells simultaneously.
2. Approach from basic research to clinical application
1) The ingestion of a Japanese poisonous mushroom Clitocybe acromelalga causes marked
reddish edema and severe allodynia (erythromelalgia) in the tips of hands and feet, which continue
for a month. Acromelic acid-A (Acro-A), a minor constituent induced tactile allodynia byintrathecal (i.t.) administration in conscious mice, which was blocked by its analogue PSPA-4.
To elucidate its suppressive effect, we synthesized 11C-labeled PET ligand and demonstrated that
PSPA-4 binds to kainate receptors in the brain and spinal cord. At present we have examined
whether there is a receptor specific to acromelic acid.
2) To alleviate postoperative pain after surgical procedures such as hepato-biliary-pancreaticmalignancies, we demonstrated that the implantation of NSAID-infiltrated gelatin hydrogel may
serve as a useful analgesic method for the long-term relief of patients after surgery.
3）To clarify pathogenesis of Huntington’s disease, we have started to analyze iPS cells preparedfrom skin cells of healthy persons and patients suffered with Huntington’s disease in collaboration
with Center for iPS Research and Application of Kyoto University..
1. 分子イメージング
1) Alzheimer 病、Parkinson 病などの神経変性疾患は脳のある部位の神経細胞死によるloss of function によることが知られている。これまで、剖検脳やモデル動物での組織標本の病理学的変化を検討されてきた。質量顕微鏡を用いて神経伝達物質の 1 つアセチルコリ
ン(ACh)の測定に成功した。質量顕微鏡の ACh の脳内分布は、ACh の分解酵素 ACh エス
テラーゼの分布と一致していた。腸閉塞・穿孔性腹膜炎を併発した症例の食餌性異物を含む病理標本パラフィンブロックを用いて、食餌性異物としてモチを同定した。病態分子イメージングセンター参加６講座が質量顕微鏡を用いた研究を推進し、徐々に成果が上がっている。
2) 侵害刺激と非侵害刺激は 1 次求心性線維により伝達され、その中枢端の脊髄で脊髄ニューロンとシナプスを形成する。脊髄後角の神経回路網の情報処理により、大脳に伝達-134-

され、痛覚と触覚と認識される。末梢組織からの感覚受容がどのように処理されるのか、
蛍光タンパクを発現する遺伝子改変マウスを用いて多光子励起顕微鏡下に、機能的、形態学的に解析を行っている。これまで、in vivo patch-clamp では１つの脊髄後角細胞の解析しかできなかったが、多光子励起顕微鏡を用いることにより多くの細胞の刺激応答を同時に見ることができるようになった。
2. 基礎研究から臨床応用へのアプローチ1) 茸毒毒ササコ Clitocybe acromelalga を食すると、1 ヶ月の長期間、四肢末端でアロデ
ィニアが生じる。その原因成分アクロメリン酸を極微量投与するとアロディニアが長期間生じ、その誘導体 PSPA-4 はアクロメリン酸のアロディニアを抑制する。抑制作用を解明するために、11C 標識 PET リガンドを合成し、脳と脊髄でカイニン酸受容体と特異的結合をすることを明らかにし、現在、アクロメリン酸に特異的な受容体があるかどうか検討している。2）肝胆膵臓癌など開腹手術後の術後痛を緩和するために、局所麻酔薬非ステロ
イド性消炎鎮痛薬(NSAID)をしみ込ませた徐放剤が有効であることを明らかにした。
さらに、3）ハンチントン病の病態を解明するために、京都大学 CiRA と健常人と患者
皮膚細胞から iPS 細胞を作製し、現在解析を進めている。
優れた成果があがった点
質量顕微鏡で神経伝達物質 ACh の測定をはじめ、病態分子イメージング参加講座との共同研究体制を構築した。
問題点
飛ばない質量顕微鏡サンプルと検出されたイオンピークの同定

2. List of Publications
Original Articles
1. Miyazaki, S., Minami, T., Mizuma, H., Kanazawa, M., Doi, H., Matsumura, S., Lu, J., Onoe,
H., Furuta, K., Suzuki, M. and Ito, S. The action site of the synthetic kainoid(2S,3R,4R)-3-carboxymethyl-4-(4-methylphenylthio)
pyrrolidine-2-carboxylic
acid(PSPA-4), an analogue of Japanese mushroom poison acromelic acid, for allodynia (tactilepain). Eur. J. Pharmacol. レフェリー有, 710, 120-127, 2013.
2. Lu, J., Katano, T., Nishimura, W., Fujiwara, S., Miyazaki, S., Okasaki, I., Aritake, K., Urade,Y., Minami, T. and Ito S. Proteomic analysis of cerebrospinal fluid before and after
intrathecal injection of steroid into patients with postherpetic pain. Proteomics レフェリー有, 12, 3105-3112, 2012.
3. Yamada, M., Yao, I., Hayasaka, T., Ushijima, M., Matsuura, M., Takada, H., Shikata, N.,
Setou, M., Kwon, AH. and Ito, S. Identification of oligosaccharides from histopathologicalsections by MALDI imaging mass spectrometry. Anal Bioanal Chem. レフェリー有, 402,
1921-1930, 2012.
4. Okuda-Ashitaka, E., Minami, T., Tsubouchi, S., Kiyonari, H., Iwamatsu, A., Noda, T.,
Handa, H. and Ito, S. Identification of NIPSNAP1 as a nocistatin-interacting protein
involving pain transmission. J Biol Chem., レフェリー有, 287, 10403-10413, 2012.
5. Araki, Y., Kaibori, M., Matsumura, S., Kwon, AH. and Ito, S. Novel strategy for the control
of postoperative pain: long-lasting effect of an implanted analgesic hydrogel in a rat model of
postoperative pain. Anesth. Analg. レフェリー有, 114, 1338-1345, 2012.
6. Sugiura, Y., Zaima, N., Setou, M., Ito, S. and Yao, I. Visualization of acetylcholine
distribution in central nervous system tissue sections by tandem imaging mass spectrometry.
Anal. Bioanal. Chem. レフェリー有, 403, 1851-1861, 2012.
7. Unezaki, S., Sasaki, A., Mabuchi, T., Matsumura, S., Katano, T., Nishio, N., Andoh, T.
Nakazawa, T. Yamamoto, T., Nakatsuka, T., Kuraishi, Y. and Ito, S. Involvement of Tyr1472
phosphorylation of NMDA receptor NR2B subunit in postherpetic neuralgia in model mice.
Mol. Pain レフェリー有, 8, 59(1-13), 2012.
8. Takagi, H., Setou, M., Ito, S. and Yao, I. SCRAPPER regulates the thresholds of long-term
potentiation/depression, the bidirectional synaptic plasticity in hippocampal CA3-CA1
synapses. Neural Plast. レフェリー有, 2012, 352829, 2012.
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Proceedings
1. Yao, I. Sugiura, Y., Zaima, N., Setou, M. and Ito, S. Visualization of acetylcholine
distribution in the brain by imaging mass spectrometry. 60th ASMS Conference on Mass
Spectrometry and Allied Topics, Vancouver, May 19-25, 2012.
2. Ito, S. Sasaki, A., Unezaki, S., Andoh, T., Matsumura, S., Katano, T., Nishio, N., Nakatsuka,T., Kuraishi, Y. and Minami, T. Characterization of postherpetic neuralgia in mice withknock-in mutation of NMDA receptor. 14th World Congress on Pain, Milano, August 27-31,
2012.
3. Yao, I., Sugiura, Y., Zaima, N., Setou, M. and Ito, S. Optimization of conditions foracetylcholine detection in the nerve tissue sections by imaging mass spectrometry 19thInternational Mass Spectrometry Conference, Kyoto, September 17, 2012.
4. Katano, T., Fukuda, M., Okumura, N., Nakazawa, T., Yamamoto, T., Takao, T. and Ito, S.
Search of neuropathic pain related proteins in neuropathic pain model mice by iTRAQ
proteome analysis. The 42nd annual meeting of the Society for Neuroscience, New Orleans,
October 13-17, 2012.
5. Yao, I., Sugiura, Y., Zaima, N., Setou, M. and Ito, S. Imaging of acetylcholine in the nerve
tissue sections with mass spectrometry. The 42nd annual meeting of the Society for
Neuroscience, New Orleans, October 13-17, 2012.
6. 矢尾育子、高雄啓三、宮川剛、伊藤誠二、瀬藤光利 Behavioral analysis of knockout
mice for F-box protein SCRAPPER. 第 33 回日本神経科学大会，第 53 回日本神経化学会大会，第 20 回日本神経回路学会大会合同大会, 神戸, 9 月 2 日, 2010.
7. 片野泰代、山崎真弥、阿部学、福田正史、奥村宣明、中澤敬信、高尾敏文、山本雅、
崎村健二、伊藤誠二 Functional analysis of neuropathic pain-related protein invivo. 第 85 回日本生化学会大会, 福岡国際会議場・マリンメッセ福岡 福岡, 12 月
14-16 日, 2012.

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Annual Report 研究成果報告書
Title of Project

Analysis of intracytoplasmic inclusions of neurodegenerative diseasesby
imaging mass spectrometory
質量顕微鏡を用いた神経変性疾患細胞内封入体の研究

P.I.

Hirofmi Kusaka, 日下博文

Affiliation

Neurology, 神経内科学講座

Collaborators

金子鋭、和手麗香、藤田賢吾、中村正孝、朝山真哉

Keywords

TGFβ,

ALS,

E-mailkusaka@hirakata.kmu.ac.jp

TDP-43

1. SUMMARY OF RESEARCH RESULTS
Background: TAR DNA binding protein of 43 kDa (TDP-43) is naturally located in the
nucleus and has been identified as the major component of cytoplasmic ubiquitinatedinclusions in patients with amyotrophic lateral sclerosis (ALS). We have reported that
TDP-43 and phosphorylated Smad2 (pSmad2), an intracellular mediator protein ofTransforming Growth Factor-(TGF) signaling, are co-localized within cytoplasmic
inclusions in the anterior horn cells of sporadic ALS patients.
Objective: To investigate the possible pathophysiological linkage between pathologic
cytoplasmic inclusions containing TDP-43 and TGF /Smad signaling.
Methods: We replicated cytoplasmic aggregates of TDP-43 in HEK293T cells bytransfecting the cells with a nuclear localization signal deletion mutant of TDP-43 andinhibiting proteasome activity, and assessed the effect of TGF/Smad signaling on thecytoplasmic aggregate formation.
Results: The aggregates contained ubiquitinated, phosphorylated, and fragmented
TDP-43, consistent with the essential features of the human pathology. Moreover, theaggregates were co-localized with pSmad2 under continuous TGFstimulation.
Overexpression of Smad2 reduced the amount of cytoplasmic aggregates in HEK293T
cells, and TGFstimulation augmented this reduction effect in a dose-dependent
manner.
Conclusion: Activation of TGF/Smad signaling system is protective against aggregate
formation of cytoplasmically mislocalized TDP-43 and may be a potential therapeutic
approach to delay progression of ALS.
我々は筋萎縮性側索硬化症(ALS)において神経細胞の生存・保護に関与する TGFβ/Smad
シグナル伝達が障害されていることをこれまでに報告してきた。本研究では、細胞モデルを用いて TGFβ/Smad シグナルと TDP-43 凝集体形成との関連について検討した。
核移行シグナル(NLS)を変異させた TDP-43 発現プラスミド(ΔNLS-TDP-43)を HEK293T
細胞に発現させると、本来核に存在する TDP-43 はプロテアソーム阻害下で細胞質内に凝集体を形成した。これらの凝集体は、リン酸化 TDP-43(pTDP-43)抗体に陽性をした。この細胞モデルを用いて、TGFβ/Smad シグナルの賦活(Smad2 過剰発現や constitutive active 型-137-

TGFβtype1 受容体(TGFCA)の共発現、外因性 TGF-β１の投与)や阻害(dominant negative 型
TGFβtype1 受容体の共発現(TGFDN)、TGF-β1 kinase inhibitor の投与)に応じて、TDP-43 凝集体形成が影響を受けるか否かについて蛍光免疫染色法やウェスタンブロッティング法
により定量的に解析した。
ΔNLS-TDP-43 と Smad2 との共発現により、不溶性画分の pTDP-43 発現量は減少し、
ΔNLS-TDP-43、Smad2 と TGFCA を共発現させると、不溶性画分の pTDP-43 発現量はさらに減少した。細胞質に pTDP-43 陽性の凝集体を持つ細胞数もそれぞれ、約 35％、約 70％の減少し、LDH 活性の測定からこの減少は凝集体陽性細胞の死滅によるものではないことが確認された。ΔNLS-TDP-43、Smad2 と TGFDN を共発現させると、TGFCA のときに認められた pTDP-43 発現量の減少効果は消失した。外因性 TGF-β１単独投与では、不溶性画分の pTDP-43 発現量に変化は認めなかったが、Smad2 との組み合わせにより、不溶性画分の pTDP-43 発現量は TGF-β1 の濃度依存性に減少効果を示した。
TGFβ/Smad シグナルの賦活により細胞死を引き起こすことなく TDP-43 凝集体形成が抑制されたことから、ALS における治療法の開発において TGF-β/Smad シグナルが重要なターゲットとなる可能性が示唆された。
優れた成果があがった点
ALS 治療法の開発において TGF-β/Smad シグナルが重要なターゲットとなる可能性が示
唆された。
問題点
今回の結果は培養細胞系であり、よりヒトの病態に近い実験系で検討する必要がある。

2. List of Publications
Original Articles
1. Nakamura, M., Kaneko, S., Ito, H., Jiang, S., Fujita, K., Wate, R., Nakano, S., Fujisawa,
J.I. and Kusaka, H.
Activation of Transforming Growth Factor-β/Smad Signaling Reduces Aggregate
Formation of Mislocalized TAR DNA-Binding Protein-43.
Neurodegener Dis. レフェリー有, 11, 2013, 182-193.

2. Nakamura, M., Kaneko, S., Wate, R., Asayama, S., Nakamura, Y., Fujita, K., Ito, H. andKusaka, H.
Regionally different immunoreactivity for Smurf2 and pSmad2/3 in TDP-43-positive
inclusions of amyotrophic lateral sclerosis.
Neuropathol. Appl. Neurobiol. レフェリー有, in press, 2012.
Proceedings
1. Nakamura, M., Kaneko, S., Ito, H., Fujisawa, J. and Kusaka, H.
Activation of Transforming Growth Factor-beta/Smad Signaling Reduces Aggregate Formationof Mislocalized TAR DNA binding prtotein-43
64th American Academy of Neurology Annual Meeting, New Orleans, USA, 2012.4.

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Annual Report 研究成果報告書
Title of Project

Elucidation of pathogenesis of psychiatric disorder
精神障害の病態研究

P.I.

Toshihiko Kinoshita, 木下利彦

Affiliation

Neuropsychiatry, 精神神経科学講座

Collaborator

齊藤幸子、Martha Shenton

Keywords

mirror neurons, diffusion tensor imaging

E-mailkinoshit@hirakata.kmu.ac.jp

1. SUMMARY OF RESEARCH RESULTS
Introduction: Deficits in attention, memory, and neuromotor functions were noted in
descriptions of schizophrenia. The purpose of the current study is to identify and to evaluate motor
related white matter integrity in patients with schizophrenia compared with healthy controls.
Method: Diffusion tensor imaging (DTI) scans were analyzed from 17 patients and 20 controls.
Mean fractional anisotropy (FA), Trace were measured from the motor related white matter.
Results: The mixed ANOVA revealed a significant main effect of group on FA of the neurons
from premotor area to putamen, indicating that schizophrenic patients had lower FA than healthy
controls (p = .002). For the FA of the neurons from primary motor area to putamen, there were nogroup effects (p = .721).
Conclusion: The primary findings of this study were that there was the groupdifference for the FA of the neurons from premotor area to putamen between theschizophrenia patients and the controls. There was no group difference for the FA of
the neurons from primary motor area to putamen. Premotor area is actively involvedin transforming sensory inputs into appropriate actions. An anterior area with mirror
neurons is located in premotor area. Our result may provide support for the theory
that schizophrenia would be associated with the abnormality of mirror neurons.
事業推進者らが研究対象とするミラーニューロンは、この社会的認知に深い関連を有すると考えられる。ミラーニューロンは、相手の行動を自分が行動するときの回路を使って処理することで理解するという脳システムの一端を担っている。「心の理論(theory ofmind)」と関連しているとも言われ、下頭頂小葉から前運動野に投射するニューロンである。
ミラーニューロンと精神分析的精神療法は関連が深い。2005 年 Gallese らによるとミラ
ーニューロンは、フロイトが提唱してきた精神療法の現場でおこる「無意識の交流」や「投影性同一視」「共感」「転移逆転移」と関連がある。
事業推進者は、精神分析の第一人者である広島市精神保健福祉センターの衣笠隆幸センター所長に精神分析的精神療法について毎週指導を受けながら、精神分析的精神療法を患者に実施している。一方で Harvard 大学医学部精神科 Psychiatry Neuroimaging Laboratory（精神画像研究所）と連携し、拡散テンソル画像解析を用いたミラーニューロンの描出に取り組んでいる。
今年度は、精神分析の第一人者の指導を週に 1～2 回受けながら精神分析的治療を患者に実施した。また、3 テスラ MRI を用いて対象患者の MRI 拡散テンソル画像を撮像した（6 名の統合失調症患者と 7 名の強迫性障害患者）。今後対照となる健常者を撮像し、一定期間の治療の後で、ミラーニューロンが変化するか調査する予定。これとは別に、ミラーニューロンに関連する統合失調症の前運動野の障害を現在まとめているところで、詳細-139-

は英文報告で示す。
優れた成果があがった点
ミラーニューロンに関連が深いとされる精神分析的精神療法の指導を受けた点。
問題点対照となる健常者の撮影がまだな点。

2. List of Publications
Proceedings
1. 齊藤幸子, 大塚達以, 浅見剛, 木下利彦, Shenton, M.
拡散テンソル画像解析によるミラーニューロンの三次元構築の試み
第 108 回日本精神神経学会学術総会, 札幌, 2012.5.

2. 齊藤幸子, 大塚達以, 木下利彦, Shenton, M.
ミラーニューロンの三次元構築の試み
第 14 回日本ヒト脳機能マッピング学会, 札幌, 2012.7.

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Annual Report 研究成果報告書

Title of ProjectPossible role of propofol’s cyclooxygenase-inhibiting property in alleviating
dopaminergic neuronal loss in the substantia nigra in an MPTP-induced murine
model of Parkinson’s disease
プロポフォールの活性化ミクログリア依存性神経細胞傷害に及ぼす影響

P.I.

Koh Shingu, 新宮興

Affiliation

Medical Control of Stress Response, 侵襲反応制御学(麻酔科学)講座

Collaborators

稲田武文

Keywords

propofol, leukotriene, dendritic cells

E-mailshingu@hirakata.kmu.ac.jp

1. SUMMARY OF RESEARCH RESULTS
Leukotrienes (LTs), divided into cysteinyl leukotrienes (CysLTs), which are important mediatorsof asthmatic responses, and leukotriene B4 (LTB4), a chemotactic and chemokinetic agent forleukocytes, are potent lipid mediators generated from arachidonic acid by 5-lipoxygenase(5-LOX). LTs are also considered to have immune-regulatory and pro-inflammatory actions.
Propofol is an intravenous anesthetic widely used for anesthesia and sedation that is alleged topossess anti-inflammatory properties. In the present study, we examined the effect of propofol on
LT production by dendritic cells (DCs). In murine bone marrow-derived DCs, propofol
significantly suppressed CysLT and LTB4 production after short-term stimulation with zymosan.
The protein levels of cytosolic phospholipase A2 and 5-LOX, or arachidonic acid release fromplasma membranes, were not affected by the presence of propofol.
Although zymosan treatment induced or enhanced the phosphorylation of ERK1/2, p-38 MAPK,
and JNK, which presumably up-regulates the activity of 5-LOX, the presence of propofol had no
additional effect on the phosphorylation status of any of these MAPKs. Similarly, zymosansignificantly increased the concentration of intracellular calcium, which is the most crucial
activator of 5-LOX, but no additional concentration changes were observed with the addition of
propofol.
Lastly, in an in-vitro cell-free ferrous oxidation-xylenol orange assay, propofol significantly
inhibited the 5-LOX activity of purified human recombinant 5-LOX enzyme with an IC50 of
approximately 7.5 M. Thus, propofol’s inhibition of 5-LOX is not likely restricted to thecircumstances surrounding the production of LTs from DCs, but applicable to other types of
immune and non-immune cells that produce LTs. The 5-LOX-inhibiting activity of propofol may,at least in part, contribute to the well-known anti-inflammatory activity of propofol.
静脈麻酔薬プロポフォールのロイコトリエン産生に及ぼす影響を、樹状細胞を用いて調べた。まずマウス骨髄より骨髄細胞を取り出し成長因子の存在下に樹状細胞を誘導した。
こうして作成された樹状細胞をザイモザンの存在下に刺激したところ、プロポフォールは濃度依存的に樹状細胞からのロイコトリエン(LT) (cysteinyl LTs, LTB4)の産生を抑制する
ことを見出した。この抑制のメカニズムを探究したところ、プロポフォールはロイコトリ
エンの前駆体である細胞膜よりのアラキドン酸の遊離には影響せず、またロイコトリエン産生酵素である 5-リポキシゲナーゼ(5-LOX)の蛋白発現や 5-LOX の核膜への移動にも影
響せず、さらには 5-LOX activating protein (FLAP)の発現にも影響を与えなかった。
さらに 5-LOX をリン酸化させ活性化させる酵素である MAP キナーゼ (ERK.・JNK・p38)のリン酸化状態を精査したところプロポフォールはいずれの酵素のリン酸化状態にも影響を与えないことが明らかになった。また同様に 5-LOX の活性化に影響を与えると考えられる細胞内カルシウム濃度を測定した結果、プロポフォールはこのカルシウム濃度にも何ら影響を与えないことがわかった。最後に、精製した 5-LOX を用いて in vitro にプロポ
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フォールの 5-LOX の酵素活性に対する影響を調べた結果、プロポフォールには直接的な
5-LOX 酵素活性抑制作用があることがわかった。
したがって、プロポフォールのロイコトリエン産生抑制作用はマウス樹状細胞に限った
ものではないことが示唆された。プロポフォールの抗炎症作用は古くからよく知られてきた事実ではあるが、われわれは今回の実験結果を通して、プロポフォールの抗炎症作用のひとつの機序として炎症物質であるロイコトリエン産生の抑制があげられることが明らかにできた。
優れた成果があがった点
静脈麻酔薬であるプロポフォールにロイコトリエン産生酵素活性抑制作用があることは今までわかっていなかった。炎症物質であるロイコトリエン産生を抑制することにより、プロポフォールが、中枢神経変性疾患や癌の進行を予防する可能性が示唆できた点で優れた成果があったのではないかと考えられる。
問題点
今回の実験は in vitro の系で得られた結果であり、実際に in vivo でもこのプロポフォー
ルのロイコトリエン産生抑制作用が再現できるかどうかが今後の問題点となりうると考えられる。

2. List of Publications
Original Articles
1. Inada, T., Ueshima, H. and Shingu, K.
Intravenous anesthetic propofol suppresses leukotriene production in murine dendritic cells.
J. Immunotoxicol, レフェリー有, in press, 2012
Proceedings

1. Ueshima, H., Inada, T., and Shingu, K.
A981 Suppression of migration of murine bone marrow-derived dendritic cells withdexmedetomidine.
Anesthesiology 2012 Annual Meeting, Washington D.C., USA, 2012.10.

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Annual Report 研究成果報告書
Title of Project

Analysis of the Brain Digitalislike Substance Underlying HYPERTENSION
高血圧の病態解明のための脳内内因性ジギタリスの局在分析

P.I.

Hakuo Takahashi, 高橋伯夫

E-mailtakahash@hirakata.kmu.ac.jp

Affiliation

Clinical Sciences and Laboratory Medicine, 臨床検査医学講座

Collaborators

小宮山豊、吉賀正亨、横井豊彦

Keywords

hypertension, endogenous digitalis, CNS, hypothalamus, sympathetic nerve

1. SUMMARY OF RESEARCH RESULTS
We have been analyzing the endogenous digitalis-like factors (EDLF) by using the liquid
chromatography / mass spectrometry (LC/MS) for many years. Since EDLFs are structurally very
similar each other with the similar molecular weight, it was found to be difficult to identify them
immunohistochemically with specific antibodies. Direct analysis of EDLFs in tissues with the
LC/MS system is impossible. Thereby, development of imaging mass spectrometry made us
possible to analyze samples like cultured cells or tissues. It is suitable particularly for analysis of
low molecular chemicals and lipids, which were difficult to detect with immunohistochemistry.
Therefore, we are aiming at the determination of the localization of EDLFs in the brain and theadrenals by the imaging mass spectrometric technology.
We have explored the possible application of EDLFs determination with authentic
marinobufotoxin and marinobufagenin. Selection of matrix to maintain samples was the mostimportant step to determine them. The lowest concentration of the authentic material to be
determined was found to be a few pg by observing the same mass spectral pattern as that of the
LC/MS. Therefore, this technology was thought to be useful for determination of EDLFs. Then,
we have tried to determine EDLFs in cultured cell samples, and found difficult to identify EDLFs
in the sample possibly because of the matrix to be used. Another reason may be the sensitivity ofthe technology because concentration of the EDLF in cell may be lower than the pg order. We aregoing to repeat experiments by using other matrix to condition the sample suitable for imaging
mass spectrometry.
従来内因性ジギタリス(EDLF)の定性的解析は免疫組織染色や液体クロマトグラフィー/
質量分析(LC/MS)を用い行われきたが、EDLF の候補物質は非常に構造式が似ており、免疫組織染色でそれぞれの物質の存在を同定するのは困難であった。従来我々の研究で用いていた LC/MS は液体サンプルの解析のため、細胞、組織内の EDLF の解析を直接行うことは困難である。しかし近年、質量顕微鏡が発達してきており、細胞、組織を直接サンプルにして従来解析が難しかった低分子化合物や脂質成分の存在を質量分析することが可能となってきている。しかし未だ質量顕微鏡を用いた EDLF の研究は報告がない。
本研究では、質量顕微鏡を用いて、われわれがすでに LC/MS を用いてマススペクトルを得ている EDLF の候補物質であるマリノブフォトキシンとマリノブファゲニンにおいて、質量顕微鏡での質量分析を行った。保持剤として用いるマトリックスの調節と標品の濃度を検討しマススペクトルを確認したところピコグラム濃度まで LC/MS と同じマスス
ペクトルを確認したことから EDLF の研究に質量顕微鏡が有用な手法となることが示唆された。
質量分析においては標品の分析がもっとも重要となるため今回の標品のマススペクト
ルの検出は質量顕微鏡を用いた細胞、組織を対象とした研究の重要な一歩となる。今後、
EDLF の分泌組織と考えられている、副腎、視床下部の培養細胞株やラットの組織をサンプルに用いて EDLF の組織および細胞内分布の検討を行い、さらに EDLF 分泌にともなう細胞膜変化を検討する予定である。
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達成度
現在、質量顕微鏡を用いて、われわれがすでに LC/MS を用いてマススペクトルを得ている EDLF の候補物質であるマリノブフォトキシンとマリノブファゲニンにおいて、質量顕微鏡での質量分析を行った。マトリックスの調節と標品の濃度を検討しマススペクトルを確認したところピコグラム濃度まで LC/MS と同じマススペクトルを確認した。しかし
質量顕微鏡は大学所有の機器を使用しているが学舎の移転にともない学内の質量顕微鏡が使用できなかった時期があり、当初の計画と比べて細胞、組織を対象とした研究への応用が遅れている。現在、副腎皮質のモデル細胞である Y-１細胞のサンプルに用いて検討をおこなっているが、現時点ではマススペクトルの検出に至っていない。
今後の計画
現在、標品においてピコグラム濃度までの検出が可能であるが、Y-1 細胞の結果から、
質量顕微鏡での検出感度を上げる必要があると思われる。そのため保持剤として用いるマ
トリックスの検討が必要と思われる。最終的には標品のフェムトグラム濃度でのマススペ
クトルの検出を目標して検討する。さらに EDLF の候補物質としてウアバインの標品のマ
ススペクトルの検出を行う。
優れた成果があがった点
Pg レベルで測定が可能であることを確認したこと。
問題点
それでも感度が足りない印象があり、精査中。

2. List of Publications
Original Articles

1. Takahashi, H.
Upregulation of the Renin-Angiotensin-Aldosterone-Ouabain System in the Brain Is the Core
Mechanism in the Genesis of All Types of Hypertension.
Inter. J. Hypertens. レフェリー有, in press, 2012

2. 高橋伯夫．
交感神経系と高血圧

呼吸と循環, レフェリー無, 60, 2012, 247-256.
Proceedings
1. 高橋伯夫．
あらゆるタイプの高血圧発症機序として中枢神経系の役割．
日本高血圧学会総会, 名古屋, 2012.9.

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Annual Report 研究成果報告書
Studies on cell treatment for complete spinal cord injury
脊髄損傷に対する細胞治療の実験的研究

Title of Project
P.I.

Toshio Nakatani, 中谷壽男

E-mailnakatant@hirakata.kmu.ac.jp

Affiliation

Spinal Cord Regeneration, 脊髄再生医学（救急医学科）

Collaborators

岩瀬正顕、前田裕仁、齊藤福樹、津田雅庸

Keywords

spinal cord injury & regeneration, transplantation, bone marrow stromal cells

1. SUMMARY OF RESEARCH RESULTS
We have been studied experimental and clinical research for the treatment of spinal cord injury.
This year, we have made our effort to summarize and publish our recent clinical work as follows:
To determine whether intrathecal administration of cultured autologous bone marrow stromal cells(BMSCs) is safe and feasible for treatment of subacute spinal injury. Five patients with complete
tetraplegia due to cervical spinal injury on admission were included. A small amount of bonemarrow was obtained during surgery for spinal fusion. BMSCs were cultured, reaching 107-108
cells. The properties and functional efficacy of the BMSCs were verified with surface marker
analysis and a neurite extension test. BMSCs were administered by lumbar puncture. The patientswere closely observed for 6 months, and the Committee on Effectiveness and Safety of ClinicalTreatment (CESCT) evaluated safety. No adverse responses were observed in biochemical andradiographic examinations. The CESCT did not recognize any harmful effects of thetransplantation, and concluded it was safe for treatment. The patients were further followed up for
1 to 4 years with no adverse responses. The recovery of American Spinal Injury Association
Impairment Scale (AIS) B and C patients at transplantation was rapid and remarkable, but gradualor limited in AIS A patients.

This study demonstrated that intrathecal administration of cultured

autologous BMSCs is safe and feasible for treatment of spinal cord injury.

The above

study report has been accepted for publication. In our next step, we are planning to administerbone marrow mononuclear cells of the patient without incubation and proliferation. We alsoplanning an experimental study to continue our clinical trials.
我々は、従来より脊髄損傷の治療に向けての実験的研究、臨床試験を行ってきた。今年
度はそれらのうち、臨床試験の成果をまとめ、公表することに精力を注いだ。
我々がこれまで行ってきた培養自己骨髄間質細胞の髄液内投与による脊髄損傷治療の安全性と実現性について検討するために、頸髄損傷による四肢完全麻痺の５症例に実施した臨床試験成績をまとめて見た。試験に於いては受傷後の頸椎固定手術に際して、少量の骨髄海綿骨を採取し、培養して間質細胞を 107 乃至 108 に増殖させ、その細胞の質に関しては表面マーカーと突起伸長試験を経て確認した後に、患者の髄液内に投与して、移植細胞を損傷脊髄に到達せしめ 6 ヶ月の試験期間、及びそれ以降も経過を追跡し、外部委員を含む効果安全性評価委員会が安全性について評価した。血液生化学検査にて、或いは画像診断にて追跡しているが、これまでのところ最長 4 年間を経過しているが、何ら骨髄細胞移植によると思われる有害反応は無く、機能的には一症例で歩行可能、一症例で装具にて起立保持可能、2 例にて車椅子の自己操縦可能となり、American Spinal Injury Association-145-

Impairment Scale にても motor score は従来の治療を行った症例に比べて、著しい改善を認めた。ただし、1 例のみは無効であった。本臨床試験は安全であり、かつ実現性が可能なものと考えている。
現在、我々は本治療法を確立するためにはより多施設での臨床試験が必要と考え、そのためには骨髄細胞を培養することなく、分離後直ちに移植する方法での臨床試験を行うべく、動物実験を経て臨床試験の準備を行っている。
優れた成果があがった点
骨髄細胞の髄液内投与の安全性に関して、有害反応が全く見られず、安全性に自信を深めた。
知覚機能は、大部分の症例で改善しているが、運動機能の改善は、受傷後 3 ヶ月を過ぎると改善が乏しいこと等が判明し、次のステップのプロトコル作りに大いに参考になったこと。
問題点
本研究は、施設基準の関係で滝井病院では細胞の分離操作を実施し出来ない。厚労大臣の許可を得て、共同研究施設である北野病院で実施している。（厚生科学審議会での審査と、北野病院の倫理委員会承認は得て居る。すべて他院の患者に対して実施しているが、本院での患者が発生した場合にも、北野病院に転院して実施する場合を考えて、念のために本学の倫理委員会にも申請した。当委員会で書類の改定を求められているうちに、10
例の予定数を終了したために、また、本院の患者は全く含まれていないために、その後の倫理委員会への再審査申請は行っていない。）
胸髄レベルの運動機能が回復したとしても、それを評価する方法が難しいこと。

2. List of Publications
Proceedings
1. 齊藤福樹, 岩瀬正顕, 中谷壽男, 津田雅庸, 前田裕仁, 宮崎秀行, 北元健, 金沢武哲, 鈴木義久, 井出千束, 福島雅典.
当院における脊髄損傷に対する再生医療への取り組み
第 40 回日本救急医学会総会, 京都, 2012.11.

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Division of Cancer

がん部門

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Annual Report 研究成果報告書
Title of Project

Dynamics of adult tissue specific stem cells by multi-color lineage tracing
method
多色細胞系譜追跡法を用いた幹細胞の維持機構の解析

P.I.

Hiroo Ueno, 上野博夫

E-mailhueno@hirakata.kmu.ac.jp

Affiliation

Stem Cell Pathology, 幹細胞病理学講座

Collaborators

比舎弘子、吉田真子、厚海奈穂、田中敏宏、駒井資弘、矢内洋次、大町太一、大江秀一

Keywords

stem cell, development, regeneration

1. SUMMARY OF RESEARCH RESULTS
Continuing last year’s research, we are trying to visualize the process of oncogenictransformation of intestinal epithelium. To this end, we are using our multi-color lineage tracing
method (rainbow mice) we developed.
It is well known that the adenomatous polyposis coli (Apc) gene is one of the key molecules(the tumor suppressor gene) for generating colon cancers. However, mutation of Apc gene canonly generate benign adenomas. Apcmin mice is one of the most popular model mice for theexperiments. In the intestinal epithelium of Apcmin mice that carry a point mutation in the Apc
gene, numerous adenomas are generated as the mice grow, and the mice eventually die because of
bleeding or obstruction of their intestine. To generate malignant tumors, we administrate several
chemicals to the Apcmin mice, and we have succeeded in generating adenomatous carcinomas. By
culturing normal and malignant intestinal epithelial cells in vitro (organoid culture), we are
currently analyzing dynamics of normal and cancer intestinal stem cells by time lapse imaging.
事業推進者はマルチカラー細胞系譜追跡法を開発して昨年度に引き続き消化管上皮細胞のがん化過程における正常成体腸幹細胞の維持機構の破綻について imaging を行っている。
大腸がんの前がん病変である大腸腺腫の発症にはがん抑制遺伝子である adenomatous
polyposis coli (Apc) 遺伝子の変異が深く関与している事が知られている。その代表的なマウスモデルの一つ、Apcmin マウスを用いる事でマウス小腸から大腸に効率的に腺腫を生じせしめる事ができる。しかしながら Apc 遺伝子変異単独では腺腫の段階で留まり、腺癌の発症にはつながらない。そこで私たちは化学発がんモデルを組み合わせ、Apcmin
マウスに起こる腺腫の一部が腺癌へと進展している状況を確認している。これらの系を私たちの開発した多色細胞系譜追跡法（レインボーマウス）と組み合わせ、大腸腺癌の起源につき解析を行い、さらに腸管上皮細胞の in vitro 培養（オルガノイド培養）の系を用いて正常およびがん化した上皮細胞培養系のイメージングを行っている。
優れた成果があがった点
正常およびがん化した腸管上皮細胞のオルガノイド培養およびレインボーマウスと組
み合わせて幹細胞を蛍光標識、オールインワン顕微鏡による Time lapse imaging により、その動態を直接観察する事が可能となった。
問題点
特に無し。

2. List of Publications
Original Articles
1. Tanaka, T., Komai, Y., Tokuyama, Y., Yanai, H., Ohe, H., Okazaki, K. and Ueno, H.

Identification of stem cells that maintain and regenerate lingual keratinized epithelial-148-

cells.
Nat. Cell Biol. レフェリー有, in press, 2013.
2. Yanai, H., Tanaka, T. and Ueno, H.
Multicolor lineage tracing methods and intestinal tumors.
JG レフェリー有, in press, 2013.
3. Zhang, H., Zheng, W., Shen, Y., Adhikari, D., Ueno, H. and Liu, K.
Experimental evidence showing that no mitotically active female germline progenitors
exist in postnatal mouse ovaries.
Proc. Natl. Acad. Sci. U.S.A. レフェリー有, 109, 2012, 12580-12585.
Proceedings

1. 上野博夫.
多色キメラ法による幹細胞の発生及び成体組織維持機構の解析
日本消化器病学会セミナー, 東京, 2012.4.
2. 上野博夫.
マルチカラーモザイクマウス（レインボーマウス）による成体幹細胞研究について
千葉大学 GCOE セミナー, 千葉, 2012.8.
3. 上野博夫.
マルチカラー細胞系譜追跡法における消化管上皮幹細胞の解析
no side GI 講演会, 大阪, 2012.9.
4. 上野博夫.
マルチカラーキメラ法を用いた幹細胞・発生研究について
金沢医科大学セミナー, 金沢, 2012.11.
5. 上野博夫.
マルチカラー細胞系譜追跡法を用いた成体組織幹細胞の同定と解析
千里ライフサイエンスセミナー, 大阪, 2013.1.
6. 上野博夫.
腸幹細胞研究の最前線〜モデルマウスを用いた大腸がん発症過程の多色細胞系譜解析
守口市医師会講演会, 大阪, 2013.5.

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Annual Report 研究成果報告書
Title of Project

Analysis of ATL development in HTLV-1 infected humanized mouse model
HTLV-1 感染ヒト化マウスを用いた ATL 発症過程の解析

P.I.

Jun-ichi Fujisawa, 藤澤順一

E-mailfujisawa@hirakata.kmu.ac.jp

Affiliation

Viral Oncology, ウイルス腫瘍学（微生物学）講座

Collaborators

竹之内徳博、田中正和、上野孝治、荀潤澤、手塚健太、姚錦春

Keywords

HTLV-1, ATL, NOG-SCID, humanized mouse, leukemia stem cells

1. SUMMARY OF RESEARCH RESULTS
Development of ATL-like symptoms has been successfully recapitulated in HTLV-1 infected
humanized mouse system that was established by intra-bone marrow transplantation of cord-blood
derived human hematopoietic stem cells into NOG mice. By using this system, we analyzed the invivo effect and its mechanism of various candidates for ATL treatment.
Treatment with the combination of anti-viral agents, zidovudine (AZT) and interferon-alpha(IFN), has been reported to be highly effective to certain type of ATL, but the mechanism of action
is totally unknown. We, therefore, examined the efficacy and the in vivo mechanism of AZT/IFN
treatment in the humanized mouse system.
HTLV-1 infected humanized mice were inoculated daily with AZT and IFN from two to fourweeks post infection and the number of infected cells and proviral loads (PVL) were analyzed.
Treatment with either AZT or IFN alone attenuated the onset of lymphoproliferative disorder in
2/3 of infected mice, while mock-treated mice were died of leukemia after 8 weeks post infection.
Furthermore, although most of treated mice retained a substantial amount of infected cells,
outgrowth of infected T-cells was kept prevented even after withdrawal of drugs. The combined
treatment suppressed the growth of infected T-cells in PBL almost completely and the PVL
remained low throughout lifetime. The suppressive effect is infected-cell specific because thenumber of uninfected human lymphocytes in PBL stayed constant on the administration of drugs.
We next examined the in vivo effect of 17-DMAG, a molecular chaperone HSP90 inhibitor,
which has been demonstrated to induce apoptosis of HTLV-1-infected cells in vitro by enhancing
the degradation of an oncogenic protein Tax of HTLV-1. Oral administration of 17-DMAG fromtwo to six weeks post infection effectively suppressed the growth of infected T-cells in peripheryand the PVL remained at the carrier level throughout lifetime. These results suggested that
infected cells with higher growth activity were more sensitive than those of lower growth,
provably due to the larger amount of Tax expression.
我々はこれまでに、ヒト臍帯血由来造血幹細胞の NOG-SCID マウスへの骨髄内骨髄移
植で作製したヒト化マウスの腹腔内に HTLV-1 産生細胞株を移入し、マウス個体内のヒト
T 細胞に HTLV-1 を感染させることで ATL 様病態を再現することに成功した。
そこで、この感染モデルを用いて、近年、一定のタイプの ATL に対する有効性が示唆されている、AZT および IFN-α を感染後 2 週〜4 週の間、連日腹腔内投与し、その効果を検証した。その結果、薬剤非投与群は感染 8 週以降、全例が白血病死するのに対し、両薬剤の単独投与群では、投与期間中、2/3 のマウスにおいて感染細胞数の増加が抑制され、-150-

その多くは投与期間後も、一定数の感染細胞の存在は認めるものの、感染細胞の増殖抑制は維持された。さらに、AZT・IFN-α 併用群では全個体で投与期間中および投与終了後も感染細胞数の増加は認められなかった。また、両薬剤の投与は非感染細胞の増殖には影響を及ぼさなかったことから、作用は感染細胞特異的であることが明らかとなった。
次に、HTLV-1 の発癌遺伝子産物 Tax の不安定化を介した HTLV-1 感染細胞の増殖抑制
効果が in vitro において示されている、シャペロン蛋白 Hsp90 阻害剤 17-DMAG の投与効果を解析したところ、感染 2〜6 週間の経口投与により、ほぼ全例の感染マウスにおいて感染細胞の増加が抑制され、投与中止後も感染細胞の異常増殖は観察されなかった。
これらの結果から、感染細胞集団の中でも、高い増殖性を示す細胞クローンが表記薬剤に対しより高い感受性を示すこと、またこの形質が Tax の発現と関連している可能性が強く示唆された。
優れた成果があがった点
HTLV-1 感染ヒト化マウスが抗 HTLV-1 あるいは ATL 発症予防薬の in vivo レベルでの評
価系として有効であることが示された。一方、これら薬剤の HTLV-1 感染細胞の増殖抑制機序を明らかにすることで、感染細胞の感染個体内増殖機序に対する理解が進むと期待される。
問題点
今後、上記薬剤によって死滅したと考えられる感染細胞群と、薬剤投与中止後に残存する感染細胞群の違いを、ウイルスおよび細胞遺伝子の発現レベルで明らかにする必要がある。

2. List of Publications
Proceedings
1. Fujisawa, J-I.
ATL-like phenotype in HTLV-1 infected humanized mouse model.
25th International Conference on Antiviral Research, 札幌, 2012.4.

2. 手塚健太, 上野孝治, 鄭真美, 荀潤澤, 田中正和, 竹之内徳博, 藤澤順一.
IBMI-ヒト化マウスを用いた HTLV-1 感染マウスモデル
第 1 回 ATL シンポジウム, 東京, 2012.8.

3. 手塚健太, 鄭真美, 上野孝治, 荀潤澤, 藤澤順一.
Carrier model of HTLV-1 infection in humanized NOG-mouse
第 71 回日本癌学会学術総会, 札幌, 2012.9.

4. 荀潤澤, 上野孝治, 齊藤峰輝, 手塚健太, 田中勇悦, 藤澤順一.
Altered pattern in viral mRNA expression of Iranian type HTLV-1 leading to enhanced
viralproduction.
第 60 回日本ウイルス学会学術集会, 大阪, 2012.11.

5. 手塚健太, 上野孝治, 鄭真美, 荀潤澤, 竹之内徳博, 藤澤順一.
HTLV-1 感染ヒト化マウスを用いた抗 ATL 治療法の評価
第 60 回日本ウイルス学会学術集会, 大阪, 2012.11.

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Annual Report 研究成果報告書
Title of Project

Identification of embryonic stem cell-like tissue committed stem cells andelucidation of their differentiation pathway: An application to cell therapy andregenerative medicine
ES 細胞様組織幹細胞の同定と分化経路の解明：細胞移植・再生医療への応用

P.I.

Yoshiaki Sonoda, 薗田精昭

Affiliation

Stem Cell Biology, 幹細胞生物学(衛生学)講座

Collaborators

佐々木豊、中塚隆介、岩城隆二

Keywords

tissue stem cells, VSEL、Escells, regemerative medicine

E-mailsonoda@hirakata.kmu.ac.jp

1. SUMMARY OF RESEARCH RESULTS
The aim of this study is to identify embryonic stem cell-like tissue committed stem cells fromvarious murine and human tissues and to apply them for the cell therapy and regenerative medicine
in the near future. Last year, we reported the identification of novel mouse dental pulp stem cell
(DPSC) and analysis of their functional properties. This year, we developed a highly effective
isolation method of mouse bone-derived very small embryonic-like stem cell (BD VSEL).
(Background) Ratajczak and his colleagues identified a unique population of very small
embryonic-like (VSEL) stem cells in adult mouse bone marrow (BM) (Leukemia 2006:20;857).
These VSELs are; 1) very small (~4 m); 2) express pluripotent stem cell markers, such as Oct4,
Nanog, SSEA-1, and Rex-1; 3); responsive to a SDF-1 gradient; 4) possess large nuclei that
contain euchromatin. It is very interesting to note that VSELs possess the potential to differentiate
into 3 germ layers in vitro and in vivo, thereby contributing to tissue/organ regeneration. These
VSELs were isolated as lineage-negative (Lin-), Sca-1-positive (Sca-1+), CD45-negative (CD45-)
cells by FACS. However, the incidence of VSELs in BM-derived mononuclear cells is ~0.01%.
Therefore, it is difficult to isolate VSELs very effectively. This study describes our recently
developed highly efficient method for isolating VSELs using enzymatic treatment of murine bone.
(Materials and Methods) Murine BM nucleated cells (BMNC) were isolated from BM
flushed from the pairs of femurs and tibiae of 8 week-old C57BL/6 mice. Erythrocytes were
removed using a hypotonic solution. Then the remaining bone tissues were thoroughly washedusing PBS- with 2% FCS. These bone tissue specimens were crushed in a mortar and thenincubated in cell dissociation buffer containing -medium with 5% FCS supplemented with 1.5
mg/ml type I collagenase and 2 mg/ml dispase at 37oC for 1 hour. Next, the BMNCs andbone-derived nucleated cells (BDNCs) were stained with various monoclonal antibodies, including
anti-lineages, anti-CD45, anti-Sca-1, anti-CXCR4, anti-CD133, and anti-PDGFR and then wereused for subsequent FACS analyses.
(Results) The R1 gate was set on the FSC channel using 4 and 10 m synthetic beads, basedon the predicted very small size of VSELs. The VSELs were isolated from BMNCs and BDNCs bymulticolor FACS, as a population of Lin-Sca-1+CD45- cells. The incidences of VSELs in the
BMNCs and BDNCs were 0.001% and 0.1%, respectively. Therefore, the enzymatic treatment ofbone tissues yielded about 100 times the efficiency for the isolation of VSELs. The bone-derived(BD) VSELs were small (< 5 m) and possessed a relatively large nucleus surrounded by a narrow
rim of cytoplasm. They expressed CD133, but not PDGFR. However, they weakly expressed
CXCR4. The gene expression profiles were analyzed using real time quantitative PCR (RQ-PCR)
to evaluate the expression of ES cell markers (Oct4, Nanog, Rex1, Dppa3), HSC (KSL) markers-152-

(c-kit, Tal1, GATA2), and MSC markers (Nestin, Ang1, CXCL12, VE-Cadherin). Unexpectedly,
BD VSELs expressed high levels of Nestin and Cadherin. However, they expressed higher levelsof Oct4 and Nanog compared to HSCs or MSCs. The gene expression profile of the BD VSELs
was clearly distinct from the well-defined populations of ES cells, KSL cells, and MSCs.
Interestingly, the number of these BD VSELs significantly increased after the induction of liver
injury by carbon tetrachloride administration. They were then most likely mobilized into theperipheral blood (PB). G-CSF did mobilize KSL cells into PB, as previously reported. However,
G-CSF did not mobilize the BD VSELs. The effects of sRANKL on the mobilization of BD VSELs
were examined in vivo. Interestingly, the number of BD VSELs significantly increased 2-3 days
after the administration of sRANKL. However, the number of VSELs in PB did not increase. These
results suggest that BD VSELs actively proliferated after liver injury and bone resorption.
(Conclusion) The present data suggest that the majority of the Lin-Sca-1+CD45- cells reside in
the bone tissue. BD VSELs resemble BM-derived VSELs. However, a RQ-PCR analysis revealed
that the gene expression profile of BD VSELs was different from those of the previously reported
BM-derived VSELs. Further studies will therefore be required to elucidate their stem cellcharacteristics and the potential relationship between BD VSELs and BM-derived VSELs.
本研究では、マウス及びヒト由来の各種組織より、ES 細胞様組織幹細胞を同定し、近未来の細胞移植・再生医療への応用を目指している。今年度は、マウス骨組織をを用いて、very small embryonic-like stem cell (VSEL)として報告されている未分化な組織幹細胞の効率的な同定・分離法の開発に成功した。
当初、既報の方法により、マウス骨髄より VSEL の分離を試みた。報告されているよ
うに、直径 5μm 程度の小型で核細胞比の大きな VSEL が分離可能であった。しかし、その採取効率は非常に低く、定量的な解析は困難であった。そこで、種々の方法を試みる過程で、マウス骨と酵素処理法などを工夫することにより、既報に比べて 50～100 倍の分離効率を有する新たな方法の開発に成功した（特願 2012-220648）。
（方法）骨髄を除去した C57BL/6 マウス骨組織を乳鉢で破砕し、酵素処理(type I
collagenase +dispase) し た 後 に 、 得 ら れ た 単 核 細 胞 を 7AAD, Annexin V, anti-lineage,anti-CD45, anti-Sca-1, anti-CD133, anti-CXCR4, anti-PDGFRa 抗体で多重染色し、FACS で
Lin-CD45-Sca-1+細胞を効率よく分取する方法を開発・確立した。本細胞を用いて以下の実験を行った。
（結果）
1）マウス骨由来単核細胞より、我々が開発した酵素処理法を用いることにより、従来の骨髄を用いる方法に比べて 50～100 倍の VSEL (Lin-CD45-Sca-1+細胞)を回収することが可能であった。
2）本細胞(以下、BD VSEL）は、直径が 5μm 程度と小型で、高い核細胞比を示した。
3）FCM 解析では、陽性マーカーとして、CD34, CD133 を発現していた。しかし、CXCR4の発現は弱かった。
4）RQ-PCR 解析では、BD VSEL は、既知の幹細胞である造血幹細胞(HSC)、間葉系幹細胞(MSC)、ES 細胞のいずれとも異なる遺伝子発現パターンを示した。興味あることに、
nestin, VE-cadherin を高発現していた。未分化細胞マーカーである Oct4, Nanog は、HSC,
MSC に比べて高く発現していた。
5）CCl4 による急性肝障害モデルでは、骨組織内で増加し、骨髄、末梢血へ動員される傾向を示した。
6）sRANKL 投与でも、骨組織内で増加傾向を示した。
7）BD VSEL は、加齢により増加傾向を示したことから、BM VSEL とは異なる in vivo
動態を示した。
（考察）
BD VSEL を効率的に分取する方法の開発に成功した。今後は、本細胞の機能解析、-153-

特に、in vitro, in vivo における組織修復の可能性について研究を進める予定である。併せて、ヒト BD VSEL に関する研究を進展させる。
優れた成果があがった点
効 率 的な マウ ス 骨由 来の BD VSEL の 同 定 ・分 離 法を 開発 し 、特 許出 願 （特 願2012-220648）した点が特筆される。
問題点
大規模な細胞移植、再生医療実験の実施には、in vitro 増幅法の開発、各種臓器障害時に置ける in vivo 動態の解明など、更なる検討が必要である。

2. List of Publications
Proceedings
1. Iwaki, R., Nakatsuka, R., Matsuoka, Y., Takahashi, M., Fujioka, T., Sasaki, Y., Asano, H.,

Uemura, Y., Kwon, A-H. and Sonoda, Y.
Development of a Highly Efficient Method for Isolating Very Small Embryonic-Like StemCells Identified in Adult Mouse Bone and their Stem Cell Characteristics.
The54th Annual Meeting of American Society of Hematology, Atlanta, USA, 2012.12.
2. 岩城隆二, 中塚隆介, 松岡由和, 高橋雅也, 藤岡龍哉, 佐々木豊, 薗田精昭.
マウス骨質由来微小幹細胞の同定・分離法の開発とその幹細胞特性
第 12 回日本再生医療学会総会, 横浜, 2013.3.
Patents
1. 特願 2012-220648
発明者：薗田精昭, 中塚隆介, 岩城隆二, 飯田寛和
出願人：関西医科大学
発明の名称：微小幹細胞の分離方法-154-

Annual Report 研究成果報告書
Title of Project

Functional analysis of overexpressing genes in tissue stem cells and relationshipwith cancer stem cells
組織幹細胞に高発現する遺伝子の機能解析とがん幹細胞との共通性
の検討

P.I.

Seiji Kanda, 神田靖士

Affiliation

Public Health, 公衆衛生学講座

Collaborators

神田靖士、下埜敬紀、岡崎はるか、王澤蘊Keywords

tissue stem cells, SP cells, cancer stem cells, pituitary, auditory pathway

E-mailkandas@hirakata.kmu.ac.jp

1. SUMMARY OF RESEARCH RESULTS
We focused on the tissue stem cells in pituitary and auditory pathway. In our previous study, we
identified the tissue stem/progenitor cells in pituitary gland and cochlear nuclei of auditory
pathway in vivo. In this study, we tried to characterized these tissue stem/progenitor cells andanalyze the gene expression.
Pituitary stem cells: Last year, we showed that the upregulated genes in SP cells were included
some specific markers of stem cells, Bcrp1 and Sca-1 etc. including a oncogene with microarray.
In order to characterize these genes’ function, we tried to clone these genes and subclone into themammalian expression vector that can control the expression of interested genes by adding
tetracycline to the culture medium. These plasmid clones were transfected to mouse iPS cells by
lipofection method. Now we tried to study the ability of differentiate and maintainance of
stemness in transfected iPS cells at several different concentrations of tetracycline in vitro. I
addition we tried to construct the new system of gene expression using viral vector instead of thatof plasmid vector.
Stem cells in Auditory pathway: Last year, we showed that about 1% of slow-cycling cells weredetected in cochlea nuclei (CN) of 3 week aged mice as SP cells, and several genes were detected
as an over-expressed genes same as common genes related with stem cells by microarray.
Especially, the related genes with neuronal differentiation such as Ndrp and neurotrohhin 3 weredetected, so we considered that these specific genes might be marker genes of tissue stem cells.
Therefore, we tried to search the tissue stem cells in different region (inferior colliculus, IC) ofauditory pathway. ICs were picked up from 1 week aged of BALB/c mice and single cellsuspension was prepared. These cells were cultured in the specific medium for neuronal stem
cells and could form the spheres after 1 week in vitro. Furthermore, we tried to form the spherefrom 1,8, 20-24, 40-48 weeks aged mice and checked the numbers of spheres. The results showed
the numbers of that was significantly decreased depend on the age compared with 1 week aged
mice. In the future, we will confirm the ability to differentiate into several cell types such asneuronal cells or other cell fate from these stem cells in vitro.
下垂体幹細胞の特徴付けと癌幹細胞との関連の検討
昨年度、下垂体幹細胞を SP 細胞を指標として単離しマイクロアレイにより発現遺伝子を探索した結果、Oncogene を含む数種類の SP 細胞に高発現する遺伝子を数種類見いだした。そこでこれらの遺伝子の機能を解析する目的でまずこれらの遺伝子をクローニング
し、テトラサイクリン存在下で発現調節可能な発現ベクターにサブクローニングした。こ-155-

れらのプラスミドをリポフェクション法でマウス iPS 細胞に遺伝子導入した。現在、テト
ラサイクリンの存在下およびその添加期間を検討することによりそれぞれの遺伝子の分化誘導能及び未分化維持能などの検討をする予定である。
聴覚伝導路における組織幹細胞の同定と特徴付け
昨年度は 3 週齢マウスの蝸牛神経核に約 1％の slow cycling cell が存在し、マイクロアレ
イの結果から未熟な細胞に共通でみられる遺伝子の発現が MP 細胞と比較して高発現していた。中でも、Ndrp や Ntf(neurotrophin)3 など neuron と分化に関連のある遺伝子が検出されたことから新規組織幹細胞のマーカーとなる可能性が考えられた。本年度は聴覚伝導路の中で異なる部位である下丘に注目し、下丘中にある幹細胞の特徴付けを行った。1 週
齢の BALB/c マウスの下丘を摘出、単細胞に分離し、特殊培地を用いて培養し sphere の形成を確認、継代を繰り返し 9th sphere まで確認した。続いて 1、8、20-24、40-48 週齢のBALB/c マウスの下丘を摘出培養し sphere の形成数を比較したところ 1 週目に比して、8
週目、20-24 週目、40-44 週目に形成される sphere 数は有意に少なく、加齢により sphere
形成能が低下することがわかった。今後、これらの幹細胞に多分化能が存在することを確認する予定である。
優れた成果があがった点
聴覚伝導路における組織幹細胞の同定を行い、その特徴付けを行った結果、蝸牛神経核の他の組織（下丘）にも組織幹細胞が存在することが示唆された。以前の報告では聴覚伝導路に神経幹細胞が存在するという報告があったが、我々は発現遺伝子の解析から、SP細胞で取得したこれらの細胞はさらに未熟な幹細胞または前駆細胞で可能性を確認する
ことができた。またいくつかの組織幹細胞のマーカーとなり得る候補遺伝子が見つけることができた。
問題点
現在、下垂体幹細胞に特異的に発現する遺伝子の機能解析を行うためマウス iPS 細胞に遺伝子導入し、その分化能などを検討している。その時に使用するベクターはテトラサイ
クリンで目的の遺伝子の発現を調節可能な系を用いている。遺伝子が導入されていることは確認できたが、目的タンパクの発現がテトラサイクリンにより制御できているかどうかがまだ確認できていない。今後、新たにウイルスベクターを用いた系を確立し、早急に分化誘導実験へと進展できるように現在進めている。

2. List of Publications
Original Articles
1. Yoshida, M., Hosomi, R., Fukunaga, K., Kanda, S. and Nishiyama, T.
Tissue Lanthanum Deposition and Phosphorus Balance in Rats with Long-term Dietary
Administration of Lanthanum Carbonate.
Biomed. Res. Trace Elements レフェリー有, 23, 2012, 40-44.
2. Tamada, Y., Kanda, S., Yoshidome, A., Hayashi, I., Miyake, M. and Nishiyama, T.
Diagnosis of active tuberculosis using MPB64, a specific antigen of Mycobacterium bovis.
Microbiol. Immunol. レフェリー有, 56, 2012, 740-747.
Proceedings
1. Okazaki, H., Kanda, S., Suzuki, H., Ooka, H., Nishiyama, T. and Tomoda, K.
Characterization of adult mouse tissue specific stem/progenitor cells in inferior colliculus.
10th Annual Meeting International Society Stem Cell Research (ISSCR), Yokohama, 2012.6.
2. 大隅泰則, 神田靖士, 大岡久司, 岡崎はるか, 西山利正, 友田幸一. 下丘における加齢にともなう GluN1 遺伝子の発現制御. 第 35 回日本分子生物学会年会, 福岡, 2012.12.
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Annual Report 研究成果報告書
Title of Project

Molecular mechanism of lymphocyte trafficking
リンパ節のホーミングの分子機構に関する研究

P.I.

Tatsuo Kinashi, 木梨達雄

E-mailkinashi@hirakata.kmu.ac.jp

Affiliation

Molecular Genetics, 附属生命医学研究所

Collaborators

片貝智哉、植田祥啓、羽廣克嘉、富山尚、安田鐘樹、近藤直幸

Keywords

chemokine, antigen receptor, integrin, cell migration

分子遺伝学部門

1. SUMMARY OF RESEARCH RESULTS
With two-photon imaging using lymph node (LN) tissues, we found that high-speed interstitialmigration of T cells required adhesion through LFA-1 and ICAM-1. Reduced lymphocyte
densities within LN augmented dependency of LFA-1 and ICAM-1 for interstitial migration of T
cells, suggesting that competitive binding to ICAM-1 is involved in T-cell intranodal migration.
To clarify the roles of Mst1 in thymocyte selection processes, we found that Mst1 deficiency
impaired negative selection against self-antigen. Two-photon imaging revealed that
CD4+thymocytes formed conjugates with Aire+ medullary thymic epithelial cells throughMst1-dependent LFA-1/ICAM-1 adhesion and recognized self-antigen. Consequently,
Mst1-deficient mice expressing OT-II and OVA transgenes exhibited defective negative selection
with increased activated effector T cells
Furthermore, Mst1 deficiency impaired antigen-specific suppression functions of regulatory T
cells. Wild-type regulatory T cells formed mobile immunological synapse formation and madefrequent contacts with antigen-presenting dendritic cells, and down-regulate a costimulatory
molecule CD86 on DC. In contrast, Mst1-deficient regulatory T cells were defective in
immunological synapse formation. The impairment of thymic selection and suppression functionsof regulatory T cells likely underlie autoimmunity development of Mst1-deficient mice.
リンパ節スライス法による 2 光子イメージングを樹立し、T 細胞の組織内高速移動には
LFA-1/ICAM-1 を介する接着が必要であることを明らかにした。リンパ節内のリンパ球密度が低下すると LFA-1/ICAM-1 依存性が高まることから、ICAM-1 に対する競合的結合がT 細胞の移動に関与していると考えられる。
胸腺細胞の選択過程において Mst1 の関与を調べた結果、Mst1 欠損によって自己抗原による負の選択が障害されていた。2 光子顕微鏡による解析の結果、胸腺組織内におけるCD4 陽性細胞は Mst1 依存性に Aire 陽性胸腺上皮細胞と LFA-1/ICAM-1 を介して接着し、抗原認識を行っていることが明らかになった。
これらの結果と一致して、OVA 抗原と OT-II
Ｔ細胞受容体の遺伝子を発現したマウスでは負の選択が障害され、末梢に活性化したエフ
ェクターＴ細胞が増加していた。
また、Mst1 欠損によって制御性 T 細胞の抗原依存的抑制作用が障害されていた。制御性 T 細胞は移動性の免疫シナプスを形成し、樹状細胞と頻繁に接触を繰り返し樹状細胞の CD86 の発現低下を引き起こしていたが、Mst1 欠損によってこれらの過程が障害されていた。したがって、胸腺および制御性 T 細胞の障害の結果、Mst1 欠損マウスは自己免疫を発症することが示唆された。
優れた成果があがった点
負の選択環境下での胸腺内自己抗原認識過程を 2 光子顕微鏡を用いて可視化でき、
Mst1 による胸腺上皮細胞との接着制御が負の選択に必要であることを示した

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問題点
特になし

2. List of Publications
Original Articles
1. Ueda, Y., Katagiri, K., Tomiyama, T., Yasuda, K., Habiro, K., Katakai, T., Ikehara, S.,
Matsumoto, M. and Kinashi, T. Mst1 regulates integrin-dependent thymocyte trafficking andantigen-recognition in the thymus. Nat. Commun. レフェリー有, 2012 3,1098.
2. Sekine, K., Kawauchi, T., Kubo, K., Honda, T., Herz, J., Hattori, M., Kinashi, T. andNakajima, K. Reelin controls neuronal migration and positioning by promoting neuronaladhesion to extracellular matrix via the inside-out activation of integrin 51. Neuron, レフェリー有, 76, 2012, 353-369.
3. Fujii, Y., Shiota, M., Ohkawa, Y., Baba, A., Wanibuchi, H., Kinashi, T., Kurosaki, T. and Baba,
Y. Surf4 modulates STIM1-dependent calcium entry. Biochem. Biophys. Res. Commun. レフ
ェリー有, 422, 2012, 615-620.
4. Harada, Y., Tanaka, Y., Terasawa, M., Pieczyk, M., Habiro, K., Katakai, T.,
Hanawa-Suetsugu, K., Kukimoto-Niino, M., Nishizaki, T., Shirouzu, M., Duan, X., Uruno, T.,
Nishikimi, A., Sanematsu, F., Yokoyama, S., Stein, J.V., Kinashi, T. and Fukui, Y. DOCK8 isa Cdc42 activator critical for interstitial dendritic cell migration during immune responses.
Blood, レフェリー有, 119, 2012, 4451-4461.
Proceedings
1. Kinashi, T., Katakai, T., Ueda, Y. and Kondo, N. Regulation of Lymphocyte “Stop and Go”:
Analysis of Lymphocyte Trafficking using Live Imaging Techniques, 第 23 回 CDB ミーティ
ング”Building multicellular systems from cellular cross-talk”, 神戸, 2013.1.
2. 木梨達雄. 接着制御破綻による自己免疫発症の機序 第 16 回中国地区小児免疫宅物療法研究会, 広島, 2013.3.
3. Kinashi, T. Mst1 regulates integrin-dependent thymocyte trafficking and antigen recognitionin the thymus. 第 41 回日本免疫学会学術集会シンポジウム, 神戸, 2012.12.
4. Katakai, T. Autotaxin/lysophosphatidic acid axis is required for the optimal interstitial T cell
migration within lymph node. 第 41 回日本免疫学会学術集会, 神戸, 2012.12.
5. Ueda, Y. Crucial roles of LFA-1 regulation in thymocyte trafficking and antigen recognition
by Mst1. 第 41 回日本免疫学会学術集会, 神戸, 2012.12.
6. Tomiyama, T. Roles of Mst1 in antigen-specific suppressor function of regulatory T cells. 第
41 回日本免疫学会学術集会, 神戸, 2012.12.
7. 木梨達雄 免疫細胞の組織内動態制御：イメージング手法による接着制御とその破綻の解析 第二回癌・免疫若手セミナー, 枚方, 2012.9.
8. 木梨達雄, 片貝智哉, 植田祥啓, 羽廣克嘉. 免疫細胞の組織内移動と停止の制御：イメージング手法による接着制御の解析 バイオイメージング学会, 京都, 2012.8.

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Annual Report 研究成果報告書
Title of Project

Generation and image analysis of a gene-modified animal model
疾患モデル動物の作製およびイメージング

P.I.

Sung-il Lee, 李成一

E-mailAffiliation

Model Animals, モデル動物部門

Collaborators

小沢まどか

Keywords

ES cells, gene-engineered mouse, dendritic cells

silee@hirakata.kmu.ac.jp

1. SUMMARY OF RESEARCH RESULTS
In previous studies, we identified small GTPase Rap1a, its effector RAPL, and Mst1 as aregulator of trafficking of a lymphocyte. These molecules control integrin activation of alymphocyte by "inside-out" signaling like a stimulus of an antigen receptor or chemokine. In our
experiment, we investigated how Rap1 signaling would be concerned in adhesion of DCs, andcontrol of trafficking using Rap1 sensor by FRET. We observed that the activation of Rap1 wasintroduced only by adhesion with ICAM-1 in BMDCs, although needs the "inside-out" signal for alymphocyte. When a stimulus of CCL21 was added, activation of Rap1 was increased, andespecially it was remarkable in leading edge. In order to investigate what kind of role Rap1
signaling has in trafficking of DCs in vivo, we labeled the Mst1+/+DCs and the Mst1-/- DCs byrespectively different fluorescence, and injected to the foot-pad of C57BL6J mice. As a result ofobserving lymph node under the 2 photon microscopes, that the efficiency of trafficking to lymph
node was decreased in Mst1-/- DCs as compared with WT DCs. It was guessed that Rap1
signaling is important for adhesion and trafficking control of DCs.
v 以前、我々は small GTPase Rap1a とそのエフェクターRAPL および Mst1 をリンパ球のTrafficking のレギュレーターとして同定した。これらの分子は、抗原レセプターやケモカ
インの刺激のような”inside-out”シグナリングでリンパ球のインテグリン活性化を制御する。本実験では、我々は DCs の接着と trafficking の制御において、FRET による Rap1 セ
ンサーを使って、Rap1 シグナリングがどのように関わっているかを調べた。
その結果、リンパ球が Rap1 を活性化するには“inside-out”シグナルが必要なのに対して、
BMDCs では Rap1 の活性化が ICAM-1 との接着だけで誘導されることを確認した。CCl21の刺激を入れた場合さらに Rap1 の活性化は上がり、特にそれは leading edge に顕著であった。
In vivo において、DCs の遊走に Rap1 シグナリングがどのような役割を持っているかを調べるために我々は Mst1+/+ DCs と Mst1-/- DCs をそれぞれ別の蛍光でラベルし、C57BL6J
マウスのフットパットへ injection した。2 光子顕微鏡でリンパ節を観察した結果、WT DCsと比較して Mst1-/- DCs はリンパ節への遊走の効率が悪いことがわかった。これらのデー
タから DCs の接着や Trafficking の制御には Rap1 シグナリングが重要であると考えられる。
優れた成果があがった点
DC の接着や Trafficking に Rap1 シグナリングが重要であると確認できた。
問題点
特になし

2. List of Publications
Proceedings
1. Ozawa, M., Ueda, Y., Lee, S.I., Katakai, T. and Kinashi, T. Roles of Rap1 signaling in
dendritic cells migration. 日本免疫学会学術集会, 神戸, 2012.12.
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Annual Report 研究成果報告書

Title of Project

Identification of causative material of small-bowel obstruction from
histopathological sections by MALDI-imaging mass spectrometry
質量顕微鏡法を用いた病理標本解析による腸管内腔閉塞原因物質の同定と発症機構の解明

P.I.

A-Hon Kwon, 権雅憲

E-mailkon@hirakata.kmu.ac.jp

Affiliation

Surgery, 外科学講座

Collaborators

山田正法、海堀昌樹

Keywords

gastric cancer, liver metastasis, ileus, imaging mass spectrometry

1. SUMMARY OF RESEARCH RESULTS
Bowel obstruction or intestinal
obstruction is a mechanical or functional
obstruction of the intestine that prevents
the normal transit of digested materials.
It can occur at any levels distal to theduodenum of the small intestine and istypically considered to represent a
medical emergency. The case in thisstudy, surgery was needed to remove thenecrotic tissue and causative materials.
Laparoscopic operation was performed toexcise the inflamed small intestine and Fig. 1 Schematic diagram of the analysis in this study
the obstructive material. For imaging MS
analysis, the surgically isolated intestinal portion and the foreign body were fixed and paraffin
embedded as performed routinely at Kansai Medical University Hospital. Even after the materials
were removed, the cause and the identity of the materials were not clarified. To obtain theinformation of the extirpated materials, imaging mass spectrometry (IMS) technique was
employed.
Direct tissue analysis using matrix-assisted laser desorption/ionization (MALDI)-mass
spectrometry (MS) provides the means for in situ molecular analysis of a wide variety of
biomolecules. This technology, IMS, allows measurement of biomolecules in their native
biological environments without the need for target-specific reagents such as antibodies. In thisstudy, we applied the IMS technique to formalin-fixed paraffin-embedded (FFPE) samples to
identify a substance(s) responsible for the intestinal obstruction caused by an unidentified foreign
body. In advance of IMS analysis, some pre-treatments were applied. After deparaffinization ofsections, samples were subjected to enzyme digestion. The sections co-crystallized with matrix
were desorbed and ionized by a laser pulse with scanning. With IMS Convolution software which
we developed, we could automatically extract meaningful signals from IMS datasets. The
representative peak values were m/z 1013, 1175, 1337, 1499, 1661, 1823, and 1985. Thus it was
revealed that the material was polymer with 162 Da unit size, calculated from the even intervals.
In comparison with mass spectra of the histopathological specimen and authentic materials, themain component coincided with amylopectin, rather than amylose. Tandem MS (MS/MS) analysis
proved that the main component were oligosaccharides. Finally, we confirmed the identification ofamylopectin by staining with periodic acid-Schiff (PAS) and iodine. These results for the first timeshow the advantages of MALDI-IMS in combination with enzyme digestion for direct analysis ofoligosaccharides as a major component of histopathological samples.
The developing rate of liver metastasis in gastric cancer is from 5 to10% and the rate of hepatic
resection is less 10%. Although the liver a frequent location of metastases from gastric cancer,
hepatic resection is seldom indicated for metastatic gastric cancer because of mutiple hepatic
nodules or associated peritoneal dissemination.
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We should analysis to detect that typical biomolecule that it is compared primary cancer tometastatic cancer with MALDI-imaging mass spectrometry.
Conclusion:
With direct MALDI-TOF MS imaging, we identified mochi as the obstructing material in asmall intestinal obstruction. The results in this study indicate that IMS is a very rapid and accurate
technique with high sensitivity for direct analysis for histopathological samples. This is the firstcase of identification of oligosaccharides as a main component of histopathological samples using
direct MS analyses. MALDI-TOF MS in combination with enzyme digestion will be a support for
the histopathological study (Yamada et al., Anal. Bioanal. Chem.402, 1921-1930, 2012).
It is difficult to collect those samples (liver metastases) in gastric cancer, because thedeveloping rate of liver metastasis in gastric cancer is from 5 to10% and the rate of hepatic
resection is less 10%.
質量顕微鏡法を用いて腸閉塞の原因となった異物の成分を同定した。
1)質量顕微鏡法を用い外科手術で摘出された異物の病理組織標本から位置情報と質量ス
ペクトルを同時取得する。
食餌性異物による腸閉塞・穿孔性腹膜炎を併発した症例に対して、食餌性異物を含む病
理標本パラフィンブロックから 4μm 厚の切片を作製し、脱パラフィン処理後、適当な消化酵素を用い、標本中の分子を切断し、前処理を標本上で行った。前処理したサンプルを質量顕微鏡法により解析した。
2)質量顕微鏡法で画像化された分子種の同定を行う。
質量顕微鏡法で得られた情報から物質の成分を推定した。ポリマーのユニット構造を予想したところ多糖類であることが予想された。更に、多段階質量分析 MS/MS 測定を用いて病理組織標本を解析した結果、グルコースをユニットとするアミロペクチンであることが判明した。これは餅の成分
図１ 質量顕微鏡法による腸閉塞原因物質解析の概要
であることが知られている（図 1）。
3)各種の特殊病理染色法を用い検証する。
病理検査で行われている各種の特殊染色法
で組織標本の染色を行った。異物の病理標本を作製し、特殊染色を施した結果、糖原か炭水化物の可能性を示唆する PAS 染色で陽性の結果を得た。S-100 蛋白染色による腫瘍検出、
Kossa 染 色 に よ る カ ル シ ウ ム 検 出 、 及 び
Dylon・Congo red でのアミロイド検出はすべ図２ 病理特殊染色結果て陰性であった（図 2）。また CD68 染色も陰性であり、生体内での異物反応は認めなかった。
以上の結果より、腸閉塞の原因物質が餅であることが明らかとなった。
4)胃癌肝転移症例におけるバイオマーカーの同定
胃癌肝転移(stageIV)は外科的適応になることは少ないが、原発・肝転移巣(H1)が遺残な
く切除できる場合には切除を施行している。胃癌再発指標となる腫瘍マーカーとしては一
般的に CEA/CA19-9 などが使用されているが必ずしも再発病変との相関を示すことがな-161-

いのが現状である。今後胃癌肝転移症例からバイオマーカーとなりうる分子を同定する予定である。
優れた成果があがった点
通常の病理検査では解明できない標本を、質量顕微鏡を使用することにより物質の成分を同定できた。腸閉塞の原因物質となった餅を質量分析を使用して分析したことが本邦で始めてであった。今回のように疾患の原因の同定はその予防に寄与できるものであると考えられる。通常の病理検査では解明できないケースに質量顕微鏡を適応することにより、原因を解明することができた。
胃癌肝転移切除症例の標本を採集しており、今後同様に分析を施行する予定である。
問題点
胃癌の肝転移に対して切除可能症例は年間 1～2 例と乏しく症例数確保に問題が残る。

2. List of Publications
1.

Yamao, J., Toyokawa, H., Kim, S., Yamaki, S., Satoi, S., Yanagimoto, H., Yamamoto, T.,
Hirooka, S., Matsui, Y., and Kwon, AH. Activation of alpha-smooth muscle actin-positive
myofibroblast-like cells after chemotherapy with gemcitabine in a rat orthotopic pancreaticcancer model. J. Hepatobiliary Pancreat. Sci. レフェリー有, 20, 206-213, 2013.

2.

Kaibori, M., Tanigawa, N., Kariya, S., Ikeda, H., Nakahashi, Y., Hirohara, J., Koreeda, C.,
Seki, T., Sawada, S., Okazaki, K., and Kwon, AH. A prospective randomized controlled trialof preoperative whole-liver chemolipiodolization for hepatocellular carcinoma. Dig. Dis. Sci.
レフェリー有, 57, 1404-1412, 2012.

3.

Araki, Y., Kaibori, M., Matsumura, S., Kwon, AH., and Ito, S. Novel strategy for the control
of postoperative pain: long-lasting effect of an implanted analgesic hydrogel in a rat model of
postoperative pain. Anesth. Analg. レフェリー有, 114, 1338-1345, 2012.

4.

Kaibori, M., Ishizaki, M., Matsui, K., and Kwon, AH. Clinicopathologic characteristics ofpatients with non-B non-C hepatitis virus hepatocellular carcinoma after hepatectomy. Am. J.
Surg. レフェリー有, 204, 300-307, 2012.

5.

Inoue, K., Nakane, Y., Michiura, T., Yamada, M., Mukaide, H., Fukui, J., Miki, H., Ueyama,Y., Nakatake, R., Tokuhara, K., Iwamoto, S., Yanagimoto, H., Toyokawa, H., Satoi, S., andKwon, AH. Ultrasonic scalpel for gastric cancer surgery: a prospective randomized study. J.
Gastrointest. Surg. レフェリー有, 16, 1840-1846, 2012.

6.

Matsui, Y., Yamaki, S., Yamamoto, T., Isizaki, M., Matsui, K., Yanagimoto, H., Toyokawa,
H., Kaibori, M., Satoi, S., and Kwon, AH. Absence of cystic duct leakage using locking clips
in 1017 cases of laparoscopic cholecystectomy. Am. Surg. 78, 1228-1231, 2012.

7.

Yokoigawa, N., Ogura, T. and Kwon, AH. Successful surgical control of peritoneal
dissemination of hepatocellular carcinoma. Case Rep Gastroenterol. レフェリー有, 6,
612-617, 2012.

8.

Yamada, M., Yao, I., Hayasaka, T., Ushijima, M., Matsuura, M., Takada, H., Shikata, N.,
Setou, M., Kwon, AH. and Ito, S. Identification of oligosaccharides from histopathologicalsections by MALDI imaging mass spectrometry. Anal. Bioanal. Chem. レフェリー有, 402,
1921-1930, 2012.
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Annual Report 研究成果報告書
Title of Project

Examination of application of IBM-BMT to intractable diseases of the nervous
system
難治性神経疾患に対する骨髄内骨髄移植の適用の検討

P.I.

Susumu Ikehara, 池原進

Affiliation

Stem Cell Disorders, 幹細胞異常症学（共同研究講座）

Collaborators

李銘、李清、石明Keywords

IBM-BMT, thymus, autoimmune diseases, tumor-bearing mice

E-mailikehara@hirakata.kmu.ac.jp

1. SUMMARY OF RESEARCH RESULTS
Gr-1 Ab administered in the IBM-BMT-plus-TT setting, suppresses tumor growth by depleting
the granulocytic myeloid-derived suppressor cells.
It has been shown that allogeneic intra-bone marrow–bone marrow transplantation (IBM-BMT)
plus thymus transplantation (TT) is effective for recipients with malignant tumors. Although TT
increases the percentage of T cells in the early period after BMT, the myeloid-derived suppressor
cells (MDSC) are still the dominant population. We used the Gr-1 Ab to deplete the granulocytic
MDSC (G-MDSC) in tumor-bearing mice that received IBM-BMT+TT Two weeks after the BMT,
the tumors in the Gr-1 Ab-injected mice were smaller than those in the control group. In addition,
Gr-1 Ab significantly increased the percentages of CD4+ and CD8+ T cells and decreased thepercentages of MDSC and G-MDSC cells. No side effects of the Gr-1 Ab on recipient or donor
thymus were observed.
担癌マウスを用い、骨髄内骨髄移植と胸腺移植と Gr-1 抗体を併用により、MDSC を除去し、癌の増殖を抑制することの検討を行った。
骨髄内骨髄移植と胸腺移植を併用するにより、悪性腫瘍の治療に有効な方法であることが明らかにした。骨髄移植後胸腺移植により、Ｔ細胞の比率は増加したが、MDSC もまた存在する。担癌マウスにおいて骨髄内骨髄移植と胸腺移植を併用し、MDSC を除去する
ために Gr-1 抗体を投与した。投与後 2 週目に腫瘍サイズは対照群より小さくなり、CD4 と CD8 の比率も増加し、MDSC の比率は低下したことが明らかにした。レシピエン
トとドナーの胸腺に Gr-1 抗体による副作用はみられなかった。
優れた成果があがった点
骨髄内骨髄移植と胸腺移植による免疫治療する上に、Gr-1 抗体を併用し、MDSC を除去することにより腫瘍の根治療法を開発した。
問題点
MDSC を除去するタイムミングと腫瘍の大きさとの関係が問題である。

2. List of Publications
Original Articles-163-

1. Li, M., and Ikehara, S. Bone marrow stem cell as a potential treatment for diabetes. J. Diabetes
Res. レフェリー有, 2013, 329596, 2013.

2. Li, M., and Ikehara, S. Bone-marrow-derived mesenchymal stem cells for organ repair. StemCells Int. レフェリー有, 2013, 132642, 2013

3. Shi, M., Li, M., Cui, Y., Liu, L., Adachi, Y. and Ikehara, S. CD4+ T cell-depleted lymphocyte
infusion impairs neither the recovery of recipient thymus nor the development of transplanted
thymus. J. Immunol. レフェリー有, 190, 2976-2983, 2013.

4. Shimo, T., Adachi, Y., Yamanouchi, S., Tsuji, S., Kimata, T., Umezawa, K., Okigaki, M,
Takaya, J., Ikehara, S. and Kaneko, K. A novel nuclear factor κB inhibitor,
dehydroxymethylepoxyquinomicin, ameliorates puromycin aminonucleoside-induced
nephrosis in mice. Am. J. Nephrol. レフェリー有, 37, 302-309, 2013.

5. Shima, C., Adachi, Y., Minamino, K., Okigaki, M., Shi, M., Imai, Y., Yanai, S., Takahashi, K.
and Ikehara, S. Neuroprotective effects of granulocyte colony-stimulating factor on
ischemia-reperfusion injury of the retina. Ophthal. Res. レフェリー有, 48, 199-207, 2012.

6. Li, M., Vanella, L., Zhang, Y., Shi, M., Takaki, T., Shapiro, JI. and Ikehara, S. Stem celltransplantation increases antioxidant effects in diabetic mice. Int. J. Biol. Sci. レフェリー有,8, 1335-1344, 2012.

7. Zhang, Y., Hosaka, N., Cui, Y., Shi, M., Li, M., Li, Q. and Ikehara, S.
Effects of intra-bone marrow-bone marrow transplantation plus adult thymus transplantation on
survival of mice bearing leukemia. Stem Cells Dev. レフェリー有, 21, 2012, 1441-1448.
Proceedings
1. 保坂直樹, 高橋伯夫, 池原進.
胸腺移植を併用した新しい骨髄移植法の悪性腫瘍への効果 II：胎生、新生、成体期の造血幹細胞と胸腺の比較.
第 101 回日本病理学会総会, 東京, 2013.4.

2. 保坂直樹, 高橋伯夫, 池原進.
骨髄内骨髄移植と成体胸腺併用移植による白血病の治療 II.
第 102 回日本病理学会総会, 札幌, 2013.6.
Patents
1. 発明の名称：悪性腫瘍の治療方法
国名：アメリカ
出願番号：786094
登録番号：US7541025
発明者名：池原 進 他-164-

Division of
Metabolic Disorders

代謝部門

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Annual Report 研究成果報告書
Study of fibril formation in the extracellular matrix
細胞外マトリックス線維形成の加齢変化に関する研究

Title of Project
P.I.

Tomoya Akama, 赤間智也

E-mailakamat@hirakata.kmu.ac.jp

Affiliation

Neuropharmacology, 神経病態薬理学（薬理学講座）

Collaborators

赤間智也、東淳子、野田和男、井上唯史、増田実希

Keywords

extracellular matrix, elastic fibers, gene-engineered mice

1. SUMMARY OF RESEARCH RESULTS
LTBP2 encodes a matrix protein, latent TGFb-binding protein 2 (LTBP-2), which is one offour LTBP family proteins (LTBP-1 to LTBP-4). LTBPs are multidomain glycoproteins containing
EGF-like domains and eight-cysteine domains, both of which are also found in other matrix
proteins, fibrillin-1 and -2. As the name indicating, LTBP bind to TGF molecules and function tokeep the signaling molecule inactive in the extracellular matrix of tissues until further induction
releases active TGF from the complex. Despite of homology and resembled domain structurewith other LTBPs, LTBP-2 does not bind to TGF molecules and thus the protein seems to have afunction distinct from direct involvement to TGF signaling. Several reports including ours
indicated association of LTBP-2 with microfibrils, which consist mainly of fibrillin-1, andsuggested to play a critical role in elastic fiber assembly. Recently homozygous mutations on
LTBP2 have been found on genetic eye diseases such as primary congenital glaucoma andmicrospherophakia in human, indicating an essential role of the protein in eye development.
Ltbp2-deficient mice have been reported as the homozygous mutant mice die at early embryonicstage, suggesting an essential role of LTBP-2 over early embryogenesis in mice. However, thisgene-knockout study of mice was inconsistent with the studies in human since LTBP2 mutation
was not reported for birth defect and/or early developmental abnormalities. Because of thisdiscrepancy, LTBP-2 seemed to have different function among species in vivo.
We have independently created conditional Ltbp2 mutant mice that have minimum alterationof mouse genome, and created systemic homozygous mutant mice to confirm their phenotype as
previously reported. Surprisingly, the homozygous mutant mice survived at adult stage without
obvious abnormality. By extensive analyses, however, we found that the homozygous mutant micedevelop lens luxation due to impaired ciliary zonule formation without typical phenotype related
to glaucoma. Suppression of LTBP2 expression in cultured human ciliary epithelial cells by
siRNA reduced formation of microfibril meshwork on the cells. Supplementation of recombinant
LTBP-2inculturemediumnot

only

rescuedmicrofibril

meshwork

formationin

LTBP2-suppressed ciliary epithelial cells, but also restored unfragmented microfibrils on ciliary
epithelial cells of Ltbp2 mutant mouse eyes under organ culture, indicating essential function of
LTBP-2 for ciliary zonule formation. LTBP-2 contained a binding domain to fibrillin-1 at a second
half domain of the entire EGF-like repeats. Mutant LTBP-2 proteins that kept the fibrillin-1
binding domain intact were not secreted from their producing cells, suggesting secretion arrest
occurred to the mutant LTBP-2 proteins due to conformational alteration led by the mutations.
Ltbp2 mutant mouse is an ideal model animal for congenital lens luxation and ectopia lentis in
human.
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細胞外マトリックスの一つである微小線維は 肺や大動脈、皮膚といった伸縮性のある組織に存在する弾性線維形成の足場になる一方で、眼球の毛様小体では微小線維そのもの
が 水晶体を毛様体筋につなぎ止める働きをしていると考えられている。微小線維は主に細胞外マトリックスタンパク質であるフィブリリン 1・2 から成り立っているが、その構築機構は未だに明らかとなっていない。我々は弾性線維および微小線維形成機構の解明に向けて研究を進めており、遺伝子改変マウスや培養細胞系を用いて線維形成に必須な遺伝
子群とその役割の解析を行なっている。
LTBP (latent TGF binding protein)蛋白質はその名の通り TGFと結合して不活性型として細胞外マトリックス中に保持し、インテグリンなどによる刺激に応じて活性型 TGFを放出すると考えられている蛋白質群である。アミノ酸配列の相同性からこれまでにLTBP1-4 の 4 種類が報告されているが、これらの中で LTBP2 だけは TGFと結合しないことが示されており、その生物学的機能が不明であった。しかしながらこれまでにいくつかの論文でヒトの遺伝病の一つである先天性緑内障の原因が LTBP2 の遺伝子変異であることが報告され、LTBP2 の眼球における機能が指摘された。また最近になって更に複数の論文で、二次的に緑内障を引き起こす遺伝性球状水晶体の患者での LTBP2 の遺伝子異常が報告された。これらの結果から LTPB2 の水晶体構造、特に水晶体と毛様体筋をつなぐ
毛様小帯と呼ばれる線維の形成への関与が強く示唆され、LTBP2 の機能不全が毛様小帯の脱落を引き起こし、さらには緑内障発症へつながると予想された。しかしこれらのLTBP2 の機能を実験的に確認するシステムが存在せず、現在のところ直接的な証明はされていない。
以前の研究でマウスにおける Ltbp2 遺伝子欠損は胎生致死と報告され、LTBP2 の発生初期における役割が示唆されていた。しかしながらこの結果はヒトの LTBP2 欠損による遺伝
病の表現型とは大きく異なっており、ヒトとマウスの種差として説明されてきた。我々はCre-loxP システムを用いた、より人為的な影響の出にくい Ltbp2 遺伝子欠損マウスを作成し、その表現型を解析した。これらの変異マウスは特に目立った表現型を示さず、野生型と同様に成体にまで正常に発生し、交配も可能であった。しかしながら詳細な解析の結果、眼球内で水晶体をつなぎ止める毛様小帯の線維形成が Ltbp2 変異マウスでは大きく損なわれており、断片化した線維が見られるのみであった。このことから Ltbp2 変異マウスはヒトの遺伝性水晶体脱臼および球状水晶体のモデル動物となり得るものと結論された。
優れた成果があがった点
我々が作成した Ltbp2 遺伝子変異マウスの表現型はヒトで発見された LTBP2 遺伝子変異による水晶体脱臼の症状とよく一致しており、ヒトの眼疾患のモデル動物となるものと考えられる。この変異マウスを用いて研究を進めることにより、水晶体脱臼発症のメカニズムを解析できるものと期待される。
問題点疾患モデル動物としてマウスを用いる場合の欠点の一つとして得られるサンプル量に限りがある点が挙げられ、特にマウスの毛様小体から得られるタンパク量は非常に少なくその後の解析に限界がある。この問題点を解決するため、正常眼球としてはブタの眼球を用いてタンパク質の調製や機能解析を行うことを検討している。

2. List of Publications
Original Articles
1. Horie, T., Baba, O., Kuwabara, Y., Chujo, Y., Watanabe, S., Kinoshita, M., Horiguchi, M.,
Nakamura, T., Chonabayashi, K., Hishizawa, M., Hasegawa, K., Kume, N., Yokode, M., Kita,
T., Kimura, T. and Ono, K.
MicroRNA-33 Deficiency Reduces the Progression of Atherosclerotic Plaque in ApoE(-/-)
Mice.
J. Am. Heart. Assoc. レフェリー有, in press, 2012.
-167-

2. Noda, K., Dabovic, B., Takagi, K., Inoue, T., Horiguchi, M., Hirai, M., Fujikawa, Y., Akama,
T.O., Kusumoto, K., Zilberberg, L., Sakai, L.Y., Koki, K., Naitoh, M., von Melchner, H.,
Suzuki, S., Rifkin, D.B. and Nakamura, T.
Latent TGFβ binding protein 4 promotes elastic fiber assembly by interacting with fibulin-5.
Proc. Natl. Acad. Sci. USA, レフェリー有, 110, 2013, 2852-2857.
3. Suzuki-Anekoji, S., Suzuki, A., Wu, S.-W., Angata, K., Murai, K.K., Sugihara, K., Akama,
T.O., Khoo, K.-H., Nakayama, J., Michiko N. and Fukuda, M.N.
In Vivo Regulation of Steroid Hormones by the Chst10 Sulfotransferase in Mouse.
J. Biol. Chem. レフェリー有, 288, 2013, 5007-5016.
Proceedings
1. 中邨智之
LTBPのTGFβ非依存的な役割
第44回日本結合組織学会学術大会・第59回マトリックス研究会大会 合同学術集会,
東京, 2012.6.
2. 赤間智也, Leona Ho, 小泉範子, 中邨智之
ジーントラップ型とノックアウト型の遺伝子変異マウスにおけるレポーター遺伝子
発現の相違
第 31 回日本糖質学会年会, 鹿児島市, 2012.9.
3. Nakamura, T.
Latent TGFβ binding protein 4 converts microfibrils to elastic fibers by interacting withfibulin-5.
Joint Meeting of the Society for Glycobiology & American Society for Matrix Biology, San
Diego, U.S.A. 2012.11.
4. Ho, L., Akama,T.O., Koizumi, N., Nakamura, T.
Disruption of B3gnt2 expression may not be achieved by gene-trapped mutant mice.
Joint Meeting of the Society for Glycobiology & American Society for Matrix Biology, San
Diego, U.S.A. 2012.11.
5. Akama, T.O., Fukuda, M., Fukuda, M.N., Nakamura, T.
Formation of an enzyme complex for efficent production of keratan sulfate
glycosaminoglycan.
Joint Meeting of the Society for Glycobiology & American Society for Matrix Biology, San
Diego, U.S.A. 2012.11.
6. 赤間智也, 福田道子, 中邨智之
β3GnT7は効率的なケラタン硫酸グリコサミノグリカン生合成に必須である
第85回日本生化学会大会, 福岡市, 2012.12.

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Annual Report 研究成果報告書
Title of Project

Analysis of signal transduction molecules involved in proliferation,
development and stemness
がん幹細胞の機能・分化に関わるシグナル分子の解明

P.I.

Satoshi Matsuda, 松田達志

E-mailmatsudsa@hirakata.kmu.ac.jp

Affiliation

Cell Signaling, 生体情報部門

Collaborators

大谷真志

Keywords

PI3K、mTOR、cell proliferation、cell differentiation

1. SUMMARY OF RESEARCH RESULTS
Members of the phosphoinositide 3-kinase (PI3K) family are important for the differentiation,
proliferation, and survival of a variety of cells, including immune cells. The basic activity of these
enzymes is to phosphorylate the third position of the hydroxyl group in the inositol ring ofphosphatidylinositol. There are four subclasses of the PI3K family; among these, class IA PI3K,
composed of a catalytic subunit (p110, , or ) and a regulatory subunit (p85, p55, p50,
p85, or p55), works downstream of receptors such as T cell receptor (TCR) and CD28 in T cells
whose signaling pathways involve protein tyrosine kinases and Ras. By using p85 and p110
double deficient mice, we found that class IA and IB PI3Ks play a redundant role in mast celldifferentiation as well as mast cell function like antigen-induced degranulation. It is thus likelythat a chemical compound inhibiting both class IA and IB PI3Ks functions as a potent anti-allergy
drug.
In many cell types, PI3K signals through mammalian target of rapamycin complex1 (mTORC1)
consisting of mTOR, Raptor, and mLST8. For instance, it has been shown that PI3K-mTORC1
axis regulates the nuclear translocation of IRF7 for the production of type I interferon (IFN) in
plasmacytoid dendritic cells. Production of IL-12 in conventional dendritic cells (cDCs) is also
regulated by molecules downstream of PI3K including mTORC1. In CD4+ T cells, thePI3K-Akt-mTORC1 axis contributes to the activation, survival, and proliferation upon stimulation
through TCR and CD28. In collaboration with Koyasu Laboratory in Keio University School of
Medicine, we found that the suppression of this axis by deletion of p85 or PI3K/mTORC1
inhibitors as well as T cell-specific deletion of Raptor, an essential component of mTORC1,
impaired Th17 differentiation in vitro and in vivo in a S6K1/2-dependent fashion. Inhibition of
PI3K-Akt-mTORC1-S6K1 axis impaired the downregulation of Gfi1, a negative regulator of Th17
differentiation. We further found that S6K2, a nuclear counterpart of S6K1, was induced by the
PI3K-Akt-mTORC1 axis, bound ROR, and carried ROR to the nucleus. These results point
toward a pivotal role of PI3K-Akt-mTORC1-S6K1/2 axis in Th17 differentiation. We also foundthat mTORC1 plays an essential role in IL-10 production from a certain type of cDCs existing in
the gastrointestinal tract, and that dendritic cell-specific loss of mTORC1 signal, which is causedby specific deletion of Raptor in dendritic cell lineage, leads to uncontrolled inflammation in theintestine. Our data reveal a new role of the mTORC1 pathway in cytokine production besides
canonical cell proliferative regulation.
PI3K-mTOR 経路は細胞の増殖・分化・生存を制御する細胞内シグナル伝達経路であり、種々のがん組織においてその異常が報告されている。本研究では、PI3K-mTOR 経路に関わる各種分子の遺伝子改変マウスを対象に、これらシグナル伝達経路の活性の増減が各種
血球系細胞の分化・増殖過程に及ぼす影響の解析を試みた。
第一に、クラス IA PI3K の制御サブユニットである p85を欠失したマウス（以下、-169-p85-KO マウス）、クラス IB PI3K の触媒サブユニットである p110を欠失したマウス（以下、p110-KO マウス）、両者を共に欠失した p85/p110二重欠損マウス（以下、DKO）
を用いて、マスト細胞機能の解析を行った。In vitro 分化系を用いた解析により、DKO マ
ウス由来の血球系幹細胞を用いた際に、明らかな分化の遅れが観察されたことから、血球系幹細胞からの効率的なマスト細胞分化誘導にクラス IA・クラス IB PI3K 経路が協調的に関わっていることが強く示唆された。さらに、分化誘導したマスト細胞を調べると、
DKO マウス由来マスト細胞の脱顆粒能が著しく低下していることが明らかとなり、クラス IA・IB の両経路を標的とすることで、効率的な抗アレルギー治療が可能になるものと考えられた。
第二に、mTOR 経路の中でも、特に細胞増殖と密接に関わる mTORC1 経路に着目し、
mTORC1 シグナルに必須のアダプター分子である Raptor 分子の細胞系譜特異的欠損マウスの樹立に取り組んだ。Raptor 分子を T 細胞系列特異的に欠失させたところ、末梢のヘルパーT 細胞の機能分化が部分的に阻害されることが分かった。また、活性化に伴う T 細胞の生存率が mTORC1 シグナルの無い状態では著しく低下する可能性も示唆され、現在、詳細を検討中である。一方、樹状細胞系譜特異的に Raptor 分子を欠損させると、腸管における従来型樹状細胞の IL-10 産生能の低下と、それに伴う腸管免疫応答の異常亢進が観察された。すなわち、mTORC1 シグナルが、従来型樹状細胞において抑制性サイトカイ
ン IL-10 の産生を介したホメオスタシス調節に関与することが明らかとなった。現在は、解析対象をさらに B 細胞系譜特異的 Raptor 欠損マウスに拡充し、解析に取り組んでいる。
優れた成果があがった点
世界に先駆けて樹状細胞特異的 Raptor 欠損マウスの樹立に成功し、従来は薬剤を用いて解析されてきた mTORC1 シグナルの重要性を、個体レベルで検証することに成功した。
問題点
キャンパスの引越に伴う保有マウスの SPF 化作業のため、年度の後半はほとんど実験を進めることができなかった。現在、T 細胞系譜特異的ならびに B 細胞系譜特異的 Raptor
欠損マウスの作出に努めており、数が揃い次第、実験の再開に取り組む予定である。

2. List of Publications
Original Articles
1. Takayama, G., Ohtani, M., Minowa, A., Matsuda, S. and Koyasu, S.
Class I PI3K-mediated Akt and ERK signals play a critical role in FcRI-induced
degranulation in mast cells.
Int. Immunol. レフェリー有, 25, 2013, 215-220.
2. Powers, S.E., Mandal, M., Matsuda, S., Miletic, A. V., Cato, M. C., Tanaka, A., Rickert, R.C.,
Koyasu, S. and Clark, M.R.
Subnuclear cyclin D3 compartments and the coordinated regulation of proliferation andimmunoglobulin variable gene repression.
J. Exp. Med. レフェリー有, 209, 2012, 2199-2213.
3. Ohtani, M., Hoshii, T., Fujii, H., Koyasu, S., Hirao, A. and Matsuda, S.
mTORC1 in intestinal CD11c+CD11b+ dendritic cells regulates intestinal homeostasis bypromoting IL-10 production.
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J. Immunol. レフェリー有, 188, 2012, 4736-4740.
4. Kurebayashi, Y., Nagai, S., Ikejiri, A., Ohtani, M., Ichiyama, K., Baba, Y., Yamada, T., Egami,S., Hoshii, T., Hirao, A., Matsuda, S., and Koyasu, S.
PI3K-Akt-mTORC1-S6K1/2 axis controls Th17 differentiation by regulating Gfi-1 expression
and nuclear translocation of ROR
Cell Reports, レフェリー有, 1, 2012, 360-373.
Proceedings
1. Matsuda, S., Nagai, S., Hoshii, T., Koyasu, S., Hirao, A. and Ohtani, M.
Role of the mTORC1 signaling pathway in acquired immunity.
第 35 回日本分子生物学会年会, 福岡, 2012.12.
2. Ohtani, M., Fujii, H., Sakai, K., Hoshii, T., Watanabe, T., Koyasu, S., Hirao, A. and Matsuda,
S.
mTOR complex 1 is critical for B cell development.
第 35 回日本分子生物学会年会, 福岡, 2012.12.
3. Nagai, S., Kurebayashi, Y., Ikejiri, A., Ohtani, M., Baba, Y., Hoshii, T., Hirao, A., Matsuda, S.
and Koyasu, S.
PI3K-Akt-mTORC1-S6K axis controls Th17 differentiation.
第 35 回日本分子生物学会年会, 福岡, 2012.12.
4. Matsuda, S. and Ohtani, M.
Role of the mTORC1 signaling pathway in T cell function.
第 41 回日本免疫学会総会, 学術集会, 神戸, 2012.12.
5. 藤猪英樹、小安重夫、松田達志
制御性 T 細胞機能発現における PI3K の役割
第 22 回 Kyoto T Cell Conference, 京都, 2012.7.
6. 永井重徳、紅林泰、大谷真志、松田達志、小安重夫
PI3K-Akt-mTORC1-S6K1/2 経路は Gfi の発現および RORγ の核移行を調節することにより Th17 分化を制御する
第 22 回 Kyoto T Cell Conference, 京都, 2012.7.
7. 大谷真志, 星居孝之, 平尾敦, 藤猪英樹, 小安重夫, 松田達志
樹状細胞における mTORC1 は IL-10 産生を介して腸管免疫に関与する
第 22 回 Kyoto T Cell Conference, 京都, 2012.7.
8. Ohtani, M., Hoshii, T., Fujii, H., Koyasu, S., Hirao, A. and Matsuda, S.
mTORC1 in dendritic cells regulates intestinal homeostasis by IL-10.
20th International Symposium on Molecular Cell Biology of Macrophages 2012, 本郷,2012.6.

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Annual Report 研究成果報告書

Title of Project

Development of novel therapeutic strategy and elucidation of functional basis
for intractable diseases by human dendritic cell research
ヒト樹状細胞によるさまざまな病態関連応答の解明と新たな難病治療戦略の開発

P.I.

Tomoki Ito, 伊藤量基

Affiliation

Internal Medicine 1, 内科学第一講座

Collaborators

野村昌作、尾形誠、中西孝尚、田中晶大、稲垣詔子、木畑佳代子

Keywords

dendritic cells, IFN-, IL-6, TNF, ICOS ligand, RANKligand, TSLP, platelets

E-mailitot@hirakata.kmu.ac.jp

1. SUMMARY OF RESEARCH RESULTS
1) Interaction between thymic stromal lymphopoietin (TSLP) and human dendritic cells (DCs)
plays an essential role in evoking inflammatory Th2 responses in allergy through expression of
CCL17 by DCs, which recruits memory Th2 cells to the skin. We newly identified the novel
cross-talk mechanism between DCs and platelets in enhancement of immune inflammatory
responses. We found that TRAP (thrombin receptor agonist peptide) induced RANK ligand
expression on platelets, which promoted the CD86 expression on DCs. We further clarified thatthe activated platelets enhanced the production of CCL17 induced by the TSLP-activated DCs
through RANK ligand. Thus, platelets have the ability to enhance the DC-mediated Th2 response
and may contribute to the allergic inflammation. Our study could provide new insights of platelet
function into the possible mechanism of DC-mediated allergic response.
2) Plasmacytoid dendritic cells (pDCs) have the potential to prime CD4+ T cells to differentiate
into IL-10-producing T regulatory cells through preferential expression of ICOS-ligand (ICOS-L).
Although pDCs produce cytokines such as type I IFNs, TNF-, or IL-6 accompanying
upregulation of ICOS-L expression during activation, the roles of the pDC-derived cytokines inT-cell priming remain largely elusive. Therefore, we investigated the functional involvement ofthese cytokines in generating IL-10-producing T regulatory cells. We found that either IFN- orIL-6 enhanced the ICOS-L-driven generation of IL-10-producing T cells from naïve CD4+ T cells
and their regulatory functions. However, IFN- stimulation in the absence of ICOS-L showed
only a marginal tendency to increase the T cell production of IL-10 and thus pDC-derived type I
IFNs could function together with ICOS-L. In addition, IL-6 functioned to generate
IL-10-producing T cells only on T-cell priming by pDCs activated by Th2 condition. Thus, type I
IFNs and IL-6 act as supplementary factors for the ICOS-L-dependent IL-10-producing T celldifferentiation in pDCs. We also showed that pDC-derived TNF- induced the ICOS-L expression
on pDCs in an autocrine manner and that IL-6 promoted the ICOS expression on T cells,
contributing to the ICOS/ICOS-L-mediated T cell response. Our results identify the molecular andfunctional basis underlying the tolerogenic pDC function in the adaptive immunity and suggestthat the ICOS-L-mediated regulatory T cell induction is backed up by the elaborate
cytokine-producing ability of pDCs.
1）アレルギー発症機序において樹状細胞（Denderitic Cell:DC）は Th2 関連アレルギー性
炎症を惹起し、アレルギー性炎症カスケードを引き起こす。上皮由来のサイトカインである胸線間質リンパ球増殖因子(thymic stromal lymphopoietin:TSLP)が、ミエロイド系 DC から TARC (thymus and activation-regulated chemokine, CCL17)を産生させることにより、メモ
リーTh2 細胞の局所浸潤とアレルギー性炎症の維持に重要な役割を果たすことが報告されている。
一方、血小板は炎症・血栓・動脈硬化において重要な役割をしており、サイトカインや-172-

TNF スーパーファミリーなどの様々な免疫メディエーターを発現している。しかしながら、血小板と DC のクロストークによるアレルギー免疫応答調節機序の報告は殆ど無い。
今回我々は新たに、活性化血小板が RANK リガンド (RANKL:Receptor Activator for
Nuclear Factor B Ligand)を発現することを同定した。この活性化血小板によって、DC の持
つ CD86 は発現増強し、さらに TSLP と同時に活性化血小板を添加することによって、DCから産生される CCL17 が増加することも明らかにした。さらに、活性化血小板に発現す
る RANKL が CD40L とともに TSLP-DC から産生される CCL17 を増加させる責任因子の一つであることを確認した。したがって、血小板は DC による Th2 免疫応答を増強し、ア
レルギー性の炎症を増幅させる可能性が示唆された。
また、活性化血小板を添加することにより成熟した TSLP-mDC とナイーブ T 細胞またはメモリーT 細胞を共培養し IL-4,IL-5,IL-13 の産生の上昇、つまり Th2 アレルギー応答に関連していることを確認した。
2）DC サブセットのうち、ヒト形質細胞様 DC (plasmacytoid DC:pDC)は、免疫応答初期には I 型 IFN 産生細胞として機能するが、その後、成熟して抗原提示細胞として機能する。
この成熟 pDC が分化誘導する T 細胞は、IL-10 産生制御性 T 細胞(Treg)であることが判明している。この機序の直接の責任分子が pDC が発現する ICOS リガンドである。一方、
pDC は ICOS リガンドの発現以外に、刺激活性化に伴い、I 型 IFN,IL-6,TNFというサイトカインを産生するが、IL-10 産生 Treg 誘導に、これらサイトカインが、どのような作用をきたすかは不明であった。
我々はこの点を検討し、pDC の持つサイトカイン産生能も、ICOS リガンドによる IL-10
産生 Treg 誘導に合理的に作動していることを明らかにした。
１． pDC が産生する I 型 IFN は、ICOS リガンド存在下で、ICOS リガンドの持つ IL-10
産生 Treg 誘導能を増強する。
２． pDC の産生する IL-6 は、Th2 環境で IL-10 産生 Treg 誘導を増強するが、Th1 環境では、T 細胞分化において IL-10 産生に寄与しない。また、IL-6 は T 細胞に対して、ICOS
発現を増強することも確認し、ICOS リガンドによる効果を増強する一因と考えられる。
３．pDC の産生する TNFは、ICOS リガンドの発現に対してもオートクラインの増強因子として機能していることを証明した。
これらの結果から pDC は抗原提示細胞としては、ICOS リガンドを発現することによりTreg 分化誘導を行うことに特化した細胞と言えるが、pDC の産生するサイトカインも、
この Treg 分化誘導を補助するように合理的に機能する。すなわち、pDC は ICOS リガン
ドを発現するのみならず、そのサイトカイン産生能も Treg 分化誘導に寄与するように精巧に作られている細胞であると考えられた。
優れた成果があがった点
DC に由来するアレルギー応答において、血小板による新たな増幅機序の可能性が考えられた。すなわちアレルギーの機序に樹状細胞のみならず、凝固系物質である血小板の役割を解明できた。「炎症」という観点に対し、樹状細胞という免疫応答の司令塔的細胞の視点と、凝固線溶系因子である血小板に着目し、その関連を解明した。血小板の多面的な効能については国内外に種々の報告があるが、樹状細胞に基点をおいた研究は類を見ず、この点も優れた成果と考えられる。
pDC が炎症免疫応答病態ではなく、むしろ末梢寛容に貢献するという報告が集積されつつあることが、pDC の持つサイトカイン産生能も、ICOS リガンドによる IL-10 産生 Treg
誘導に合理的に作動していることを明らかにし、pDC の持つ Treg 分化誘導能の細胞学的分子メカニズムを解明できた。
問題点
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in vivo での活性を検討できていないので、今後、マウスを用いた系での生体内での検討が必要と思われる

2. List of Publications
Original Articles
1. Shimamoto, K., Ito, T., Ozaki, Y., Amuro, H., Tanaka, A., Nishizawa, T., Son, Y., Inaba, M.
and Nomura, S. Serum interleukin-6 before and after therapy with tocilizumab is a principal
biomarker in patients with rheumatoid arthritis. Rheumatology. レフェリー有, , in press,
2013.
2. Ogata, M., Ito, T., Shimamoto, K., Nakanishi, T., Satsutani, N., Miyamoto, R. and Nomura, S.
Plasmacytoid dendritic cells have a rational cytokine-producing capacity to enhance ICOS
ligand-mediated IL-10 production during T-cell priming. Int. Immunol. レフェリー有 25,
2013, 171-182.

3. Ito, T. Immune crosstalk between platelets and dendritic cells. J. Japanese Society forLaboratory Hematology レフェリー有, 13, 2012, 397-340.
4. Ozasa, R., Hotta, M., Yoshimura, H., Nakanishi, T., Tamaki, T., Fujita, S., Nakamichi, N.,
Miyaji, M., Ishii, K., Ito, T. and Nomura, S. Therapeutic effect of bortezomib for primary
plasma cell leukemia followed by auto/allo stem cell transplantation. Int. Med. Case Rep. J.
レフェリー有, 5, 2012, 39-43.
5. Conrad, C., Gregorio, J., Wang, Y-H., Ito, T., Meller, S., Hanabuchi, S., Anderson, S.,
Atkinson, N., Ramirez, P. T. Liu, Y-J., Freedman, R. and Gilliet, M. Plasmacytoid dendritic
cells promote immunosuppression in ovarian cancer via ICOS co-stimulation of Foxp3+ T
regulatory cells. Cancer Res. レフェリー有, 72, 2012, 5240-5249.
6. Fujita S, Nakanishi T, Yoshimura H, Hotta M, Nakamichi N, Tamaki T, Ishii K, Ito T,
Nomura S. TGFβ(1) and sCTLA-4 levels are increased in eltrombopag-exposed patients with
ITP. Thromb. Res. レフェリー有, 130: 2012 415-9.
Books
1. 伊藤量基 反応する核酸の種類と樹状細胞サブセットの産生するサイトカイン 科学評
論社, 臨床免疫・アレルギー科, 2012, 10 ページ, 57, 234-243.

2. 伊藤量基 樹状細胞の分化・機能発現とサイトカイン 科学評論社, 臨床免疫・アレル
ギー科特別増刊号, 2012, 9 ページ, 57 suppl.21 689-697.
3. 伊藤量基 形質細胞様樹状細胞からの I 型インターフェロン産生抑制による新たな自己免疫疾患の治療 科学評論社, 臨床免疫・アレルギー科, 2012, 8 ページ, 58, 217-224.
Proceedings
1. 伊藤量基 血小板と樹状細胞の免疫クロストーク 第 13 回日本検査血液学会, 大阪,
2012.7.

2. 中西孝尚 An intimate link between dendritic cells and platelets in the maintenance of allergic
response. XXVII Congress of the International Society for Advancement of Cytometry, ドイ
ツ・ライプツィヒ, 2012.6.
3. 中西孝尚 樹状細胞と血小板の共同作用によるアレルギー性炎症の新たな機序 第 22回日本サイトメトリー学会, 大阪, 2012.6.

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Annual Report 研究成果報告書
Title of Project

Invention of cardiac regeneration therapy using molecular imaging techniques
分子イメージングによる心筋再生療法の開発

P.I.

Ichiro Shiojima, 塩島

Affiliation

Internal Medicine 2, 内科学第二講座

Collaborators

岩崎真佳、藤高啓祐、秦亮嘉

Keywords

cardiac stem cells, embryogenesis, myocyte regeneration

一朗

E-mailshiojima@hirakata.kmu.ac.jp

1. SUMMARY OF RESEARCH RESULTS
The heart is the first organ to form during embryogenesis, and its contractile function is
maintained throughout life. Since cardiac myocytes lose their ability to proliferate soon after birth,the heart has been recognized as a post-mitotic organ that does not regenerate in response tovarious external stimuli. Recent studies however support the notion that small number of cardiac
myocytes are continuously renewed and that there exist cardiac intrinsic stem cells in the adult
heart that are capable of differentiation into various cell types including cardiac myocytes,
endothelial cells, and smooth muscle cells. The mechanism that controls the cardiac stem cell self
renewal and differentiation is largely unknown and, if elucidated, will have a broad impact in thefield of stem cell biology, organogenesis, and tissue regeneration.
We previously reported that human cardiac stem-like cells that are capable of differentiating
into multiple cell types in the heart can be mobilized into circulation during open heart surgery.
The specific aims of the present study are three-fold:(1) Establishment of the methodology to mobilize human cardiac stem cells with high efficiency
into circulation(2) Identification of cardiac stem cell-specific surface markers that can be used for imaging ofcardiac intrinsic stem cells(3) Identification of stem cell niche components in the heart
In this year we focused on the establishment of the methodology to mobilize human cardiac
stem cells with high efficiency into circulation and found that intravenous infusion of heparinmobilizes cardiac stem cells in peripheral circulation. Mechanistically, serum concentration ofhepatocyte growth factor (HGF) was markedly upregulated following heparin infusion in humansubjects, and HGF treatment was able to mobilize cardiac stem cells in rats, suggesting that
heparin induces the mobilization of cardiac stem cells via upregulation of HGF.
The observation that a relatively simple method (i.e., heparin infusion) is highly effective tomobilize cardiac stem cells is of both basic and clinical importance. Using this method, we willproceed to the identification of human cardiac stem cell-specific surface markers that can be used
for imaging of cardiac intrinsic stem cells. Identification of cardiac stem cell-specific markers willfurther advance our understanding of cardiac homeostasis and regeneration.
心臓は胎児発生の初期に形成され、その後も生涯にわたりその機能が維持される。哺乳類では心筋細胞の分裂能が出生直後に失われることから、心臓は再生しない臓器であると従来考えられてきたが、近年の解析により、成人期の心臓に心筋細胞や血管系の細胞に分化しうる組織幹細胞が存在することや、正常なヒトにおいても年間約１％の心筋細胞が新たに作られた細胞と入れかわることなどが明らかにされた。しかしながら組織幹細胞による心臓再生の分子メカニズムの詳細については現在ほとんど知られておらず、その制御機-175-

構が明らかになれば、幹細胞分化・組織の恒常性維持・器官再構築といった点において大きなインパクトを持つものと考えられる。
我々はこれまで心筋細胞・血管内皮細胞・血管平滑筋細胞など心臓内の複数の細胞種に分化しうるヒト心臓組織幹細胞が心臓手術中に末梢血に動員されることを明らかにしてきた。そこで本研究では以下の３点について検討することを目的とする。
①ヒト心臓幹細胞を効率よく末梢血中に動員する方法の確立②ヒト心臓幹細胞のイメージングに使用可能な特異的表面抗原の同定③心臓組織において心臓幹細胞が局在するニッチの同定
平成 24 年度の研究により、ヘパリンの静脈投与がヒト心臓からの組織幹細胞動員を起こ
すこと、また、ヘパリンが細胞外マトリクスに結合した hepatocyte growth factor (HGF)を遊離させ HGF 血中濃度を上昇させることが、心臓組織幹細胞が末梢血へ動員される機序であることが明らかになった。
優れた成果があがった点
ヒト心臓組織幹細胞が血中へ動員される機序を明らかにし、効率よく in vitro で培養することが可能になった点が最も優れた成果であると考えられる。
問題点
今後詳細な解析を進めていく上では動物モデルの確立が必須である。現在マウスやラットを用いてヘパリンによる心臓幹細胞の末梢血への動員を試みているが、ヒトの場合と異なり一定数の幹細胞が再現性をもって得られるところまでには至っていない。今後実験条
件のさらなる検討が必要であると考える。

2. List of Publications
Proceedings
1. 藤高啓祐.
ヘパリンによる多能性ヒト Mesoangioblast の循環血中への動員
日本再生医療学会, 横浜, 2012.6.

2. Hata, Y.
Multipotent human mesoangioblast mobilization induced by heparin.
European Society of Cardiology Congress, ドイツ・ミュンヘン, 2012.8.
3. Hata, Y.
Heparin induces the mobilization of human multipotent circulating mesoangioblasts during
cardiac catheterization.
日本循環器学会, 横浜, 2013.3.

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Title of Project

Molecular imaging in the development of carcinogenesis and tissue
regeneration in inflammatory diseases of the digestive organs.
炎症性消化器疾患の発癌と再生における分子イメージング解析

P.I.

Kazuichi Okazaki, 岡崎和一

E-mailokazaki@hirakata.kmu.ac.jp

Affiliation

Internal Medicine 3, 内科学第三講座

Collaborators

西尾彰功、内田一茂、坂口雄沢、福井由理、栗島亜希子、住本貴美、光山俊行、岡崎敬、豊永貴彦、福井寿朗、吉田勝則、岸本房久、高橋悠、
鈴木尚、山口貴志

Keywords

molecular imaging, stem cells, tumor, epithelial regeneration, tumor, colitis,
autoimmune pancreatitis

1. SUMMARY OF RESEARCH RESULTS
In the pancreas and gastrointestinal tract, we studied the significance of IgG4-positive cells andpSmad2/3L-Thr immunostaining-positive cells as biomarkers in inflammatory gastrointestinal
disorders. In differenciating IgG4-related autoimmune pancreatitis and pancreas cancer, thenumbers and ratios of Tregs, IgG4-positive, and IgG-positive cells immunohistochemically stainedwith anti-Foxp3, IgG4, and IgG antibodies, respectively, were useful. In the regenerative study,
pSmad2/3L-Thr immunostaining-positive cells were detected in specific epithelial cells around
crypt bases of both small intestines and colons of C57BL/6 mice, where respective putative stem
cells are thought to exist. In small intestines, most pSmad2/3L-Thr immunostaining-positive cells
were located around the 4 cell position from the bottom of crypts (just above Paneth cells). A few
of them were also located at the places for CBCs (between Paneth cells). Double
immunofluorescent staining for pSmad2/3L-Thr and Ki67 demonstrated that pSmad2/3L-Thr
immunostaining-positive cells were located adjacent to actively proliferating cells (proliferatingzone) in both small intestines and colons, but immunohistochemical co-localization of
pSmad2/3L-Thr with Ki67 was never observed. In the regeneration phase of DSS-induced colitis,
pSmad2/3L-Thr immunostaining-positive cells significantly increased, concurrent withhyperplastic changes and an increase in the number of Ki67 immunostaining-positive cells.
Double immunofluorescent staining for pSmad2/3L-Thr and CK8 demonstrated that
pSmad2/3L-Thr immunostaining-positive cells were epithelial cells, but that for pSmad2/3L-Thrand Chr-A demonstrated that they were not enteroendocrine cells. In higher magnification with alight microscope, pSmad2/3L-Thr immunostaining-positive cells indicated undifferentiatedmorphological features and were reconfirmed around crypt bases. pSmad2/3L-Thr
immunostaining-positive cells showed co-localization with CDK4 immunostaining-positive cells.
Although pSmad2/3L-Thr immunostaining-positive cells were included in the
immunostaining-positive areas of MSI-1, they showed different localization from DCAMKL1
immunostaining-positive cells. MSI-1 and DCAMKL1 are candidate biomarkers for epithelial
stem (or progenitor) cells in the small intestine and colon. Most pSmad2/3L-Thr
immunostaining-positive cells were included in the Lgr5-GFP positive cells in crypt bases ofcolons. Conversely, in small intestines, most pSmad2/3L-Thr immunostaining-positive cells
showed different localization from Lgr5-GFP positive cells in the places for CBCs and werearound the 4 cell position, although a few of them were included in the Lgr5-GFP positive cells.
BrdU label-retaining assay revealed that pSmad2/3L-Thr immunostaining-positive cells wereslow-cycling cells (i.e., LRCs).
1． 炎症性消化器疾患における病態解析とバイオマーカー探索：
IgG4 関連自己免疫性膵炎(AIP)と膵癌における免疫担当細胞と IgG4 陽性細胞の関連性-177-

の検討をおこない、膵癌上流閉塞性膵炎においても IgG4 と Foxp3 の相関関係が認められた(R＝0.733)ことより膵癌周囲の膵炎と AIP で IgG4/IgG 陽性細胞比率および IgG4 陽性細胞数の解析は病態解析のバイオマーカーになる可能性が示唆された。
2. 消化管・膵・肝再生における幹細胞の探索と粘膜再生・発癌メカニズムの解析：
最新の幹細胞、細胞周期研究の知見や、各消化器疾患モデルマウスの知見を応用することにより、消化管粘膜上皮・膵における有用な新規組織幹細胞マーカーとしてリンカー部
スレオニンリン酸化 Smad2/3 蛋白(pSmad2/3L-Thr)を同定した。正常マウス小腸・大腸粘膜や DSS 起因性大腸炎モデル、IL-10 ノックアウトマウス大腸炎・大腸癌モデル・セルレ
イン膵炎といったモデルを作製し、pSmad2/3L-Thr と Ki-67 の蛍光二重免疫染色法にて同様に組織幹細胞を検出し、組織上皮幹細胞のマーカーとして利用できることを確認した。
特に DSS 起因性大腸炎モデル・セルレイン膵炎については pSmad2/3L-Thr 強陽性細胞を粘膜障害・再生の経過とともに経時的に確認し、活動性病変部では pSmad2/3L-Thr 強陽性幹細胞はほぼ消失しており、再生治癒状況が進行するにつれ消化管では粘膜基底部と膵では tubular complex における pSmad2/3L-Thr 強陽性幹細胞は著明に増加していた。消化管
粘膜には二種類(slow- or rapid- cycling)存在するとされる組織幹細胞のうち、slow-cyclingstem cell の特徴に合致すると考えられた。IL-10 ノックアウトマウス大腸炎・大腸癌モデルにおいては pSmad2/3L-Thr 強陽性細胞の分布を腸炎部、癌部にて観察したが、粘膜が過形成となる腸炎部では pSmad2/3L-Thr 強陽性細胞が著増していたが、癌部と思われた病変部では pSmad2/3L-Thr 強陽性細胞はほとんど発現していなかった。内視鏡的に摘出された組織標本(正常消化管粘膜組織や各消化管腫瘍組織)を利用して、同様に pSmad2/3L-Thr 強陽性細胞を検出する実験を予備的に施行したところ、腫瘍部において pSmad2/3L-Thr 強陽性細胞が散在性に認められた。
優れた成果があがった点
1． 膵癌において IgG4 陽性細胞の検討はいくつか報告があるがと IgG4 関連自己免疫性膵炎との比較で詳細に検討したものはなく、臨床上問題となる限局性を呈する自己免疫性膵炎と膵癌の鑑別において非常に有意義な研究となった。
2． 消化管粘膜上皮・膵における有用な新規組織幹細胞マーカーとしてリンカー部スレオ
ニンリン酸化 Smad2/3 蛋白(pSmad2/3L-Thr)を同定することができた。消化管粘膜上皮幹細胞のうち、pSmad2/3L-Thr 陽性幹細胞は、slow-cycling stem cell の特徴に合致し、細胞周期 G0 の状態にあり、細胞増殖周期に入っていく直前の状態の幹細胞を捉えていると考えられた。
問題点
1． 難病を対象としており、十分なサンプル数の確保が容易でない。
2． 細胞内蛋白質のリン酸化部位特異的な発現を見ているため、遺伝子を lineage tracing
するような遺伝子発現の実験と結びつけることが出来ず、また生きたまま細胞を回収できないためその後の培養実験などをすることが出来ない点。

2. List of Publications
Original Articles
1. Fukui, Y., Uchida, K., Sumimoto, K., Kusuda, T., Miyoshi, H., Koyabu, M., Ikeura, T.,
Sakaguchi, Y., Shimatani, M., Fukui, T., Matsushita, M., Takaoka, M., Nishio, A., Shikata, N.,
Sakaida, N., Uemura, Y., Satoi, S., Kwon, AH. and Okazaki, K.
The similarity of Type 1 autoimmune pancreatitis to pancreatic ductal adenocarcinoma withsignificant IgG4-positive plasma cell infiltration.
J. Gastroenterol. レフェリー有, in press, 2012.
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Proceedings
1. 内田一茂, 福井由理, 楠田武生, 三好秀明, 小藪雅紀, 池浦司, 島谷昌明, 高岡亮, 岡崎
和一.
膵癌における IgG4 陽性形質細胞の検討
第 110 回日本内科学会講演会, 東京, 2013.4.

2. 高橋悠, 福井寿朗, 岡崎和一.
pSmad2/3L-Thr の消化管幹細胞マーカーとしての検討とその応用
第 99 回日本消化器病学会総会, シンポジウム 14「消化器領域における幹細胞研究の進歩」, 鹿児島市 2013.3.
3. 高橋悠, 福井寿朗, 岡崎和一.
消化管上皮における pSmad2/3L-Thr 陽性細胞の検討
第 98 回日本消化器病学会総会, シンポジウム 2「消化管 stem cell の新たな展開」, 東京, 2012.4.
4. Takahashi, Y., Fukui, T., Suzuki, R., Kishimoto, M., Uchida, K., Matsuzaki, K., Nishio, A. andOkazaki, K.
The specific linker phosphorylation of Smad2/3 indicates epithelial stem cells in oesophagus.
20th United European Gastroenterology Week. Amsterdam, Netherlands, 2012.10.
5. Fukui, T., Suzuki, R., Takahashi, Y., Kishimoto, M., Uchida, K., Nishio, A. and Okazaki, K.
The specific linker phosphorylation of Smad2/3 indicates epithelial stem cells in colon;
particularly increasing in the regeneration phase of murine model of dextran sulfate
sodium-induced colitis.
GI Research Academy 2012, Tokyo, 2012.6.
6. Takahashi, Y., Fukui, T., Kishimoto, M., Nakajima, A., Sakaguchi, Y., Uchida, U., Nishio, A.,
Matsuzaki, K. and Okazaki, K.
The Specific Linker Phosphorylation of Smad2/3 Indicates Epithelial Stem Cells in
Esophagus.
Digestive Disease Week 2012, San Diego, USA, 2012.5.

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Annual Report 研究成果報告書

Title of Project

Clarification of skin and soft tissue aging and tissue regeneration andapplication in diagnosis and treatment using molecular imaging.
皮膚・軟部組織の加齢変化、組織再生における分子イメージングによる病態解明と診断治療への応用

P.I.

Kenji Kusumoto, 楠本健司

Affiliation

Plastic and Reconstructive Surgery, 形成外科学講座

Collaborators

鈴木健司、森本尚樹、堀尾修、日原正勝、覚道奈津子、田中義人、光井俊人、畔熱行、笹尾卓史、櫛田哲史、勝部元紀、三宅良平、上田実幸、
堀内あい、プリシラ、小倉常敬、原朋也
Regeneration, reconstruction, wound healing, tissute, cell, skin, fat, bone,cartilage, nerve, vessel

Keywords

E-mailkusumoto@hirakata.kmu.ac.jp

1. SUMMARY OF RESEARCH RESULTS
In our Department, improvement and healing after various growth factors applications promotewound healing is evaluated in biological and pathological wound healings by pathologicalmolecular imaging procedures. Pathological molecular procedures contributes basic research ofwound healing and regenerative medicine to treatment and reconstruction in medical fields.
In our research of 2012, bone regeneration, adipose-derived stem cells, and platelet-richplasma are in main studied.
Bone regeneration: Once critical sized defects of rat calvarial bone was produced, bonemorphogenetic protein (BMP), hydroxyapatite (HAP), and the mixture (BMP/HAP) arecomparatively implanted. the mixed BMP/HAP group showed the highest level of bone induction.
And osteoinduction using BMP has been studied in osteoporosis rats.
Adipose-derived stem cells (ASCs): Multi-potentiality of ASCs in differentiation is clarified at
the basis of differentiation into bone, cartilage, and fat tissue. It is confirmed that ASCs is possible
to contribute to regenerate broad medical fields.
Platelet-rich Plasma (PRP): Relation between PRP application and αSMA is clarified. On
wound healing models, PRP contributes vascularization. In chronic wounds, pressure sores andincurable wounds, PRP promotes wound healing so much.
形成外科学講座では、創傷の生理的ならびに病的治癒過程を病態分子イメージングでとらえ、さらに種々の組織再生因子の負荷により病的創傷治癒が生理的創傷治癒に近づける
過程を病態分子イメージングとしてとらえることで、明らかな改善、治癒に関与するエビ
デンスを出すことを目指している。この病態分子イメージングにより創傷治癒、組織再生における基礎研究を治療学、再建学につなげる大きな役割を果たすものと考えている。
平成 23 年度は、骨再生、脂肪組織由来幹細胞、多血小板血漿(PRP)で、それぞれの成果を挙げた。
骨再生：ラット頭蓋骨の規格化した限界的骨欠損を作成し、骨形成タンパク(BMP)単独、あるいは hysroxyapatite(HAP)単独、両者併用で埋入した。この結果、骨欠損部の修復再生が生じるが、これを X 線画像、組織学的所見での画像解析を行った。これにより、BMP
が HAP より優れ、BMP+HAP 併用が最も骨欠損修復再生に効果的であることが明らかとなった。
脂肪組織由来幹細胞(ASCs)

細胞内に低分子量 G タンパク質 Rho が存在するが、exoenzyme C3 transferase が Rho inihibitor である。今回、C3 を加えることによる ASC
cytoskeleton の変化を観察した。C3 負荷によりストレスファイバーや α-SMA の発現も抑制された。C3 は ASCs の細胞内伝達の抑制因子である可能性が示唆された。
多血小板血漿(PRP) 均一採皮創で、PRP をハーフサイドで上皮化を比較検討した。術後13 日目に組織生検を行い PRP 群で、有意に表皮が厚く、血管数と αSMA が多かった。PRPの有意なる創傷治癒促進効果と判断した。また、PRP は有意に αSMA の増加と瘢痕拘縮-180-を生じることを明らかにした。
優れた成果があがった点＃骨再生で、BMP と HAP 併用が有用、有効であることが明らかできたこと＃ASCs において、多分化能により、再生の基盤となることが示唆されたこと＃PRP による αSMA の増加と瘢痕拘縮の関連と創傷治癒促進の結果が明らかとなったこと
問題点
BMP、ASCs、PRP の更なる探求と、臨床応用での投与法の追究が今後の課題である。

2. List of Publications
Original Articles
1. Notodiharjo, F.Z., Kakudo, N., Kushida, S., Suzuki, K. and Kusumoto, K. Bone regeneration
with BMP-2 and hydroxyapatite in critical-size calvarial defects in rats. J. Cranio-maxillo-fac
Surg. レフェリー有, 40, 2012, 287-291.

2. 田中義人, 楠本健司, 竹本剛司, 日原正勝, 田中寧子 当院開院から現在までのケロイド 135 症例の検討 瘢痕･ケロイド治療ジャーナル, レフェリー有, 6, 2012, 66-68.
3. 楠本健司, 田中義人, 山本純 傷跡・瘢痕拘縮治療の理論と実際 5)組織拡張器(ティッシュ・エキスパンダー)の適応と使用 形成外科, レフェリー有, 55, 2012, S340-S344.
4. 堀尾 修, 楠本 健司 血管腫・血管奇形の分類と診断 形成外科 , レフェリー有, 55,
2012, 1169-1176.
5. Kakudo, N., Kushida, S., Ogura, T., Hara, T., Suzuki, K. and Kusumoto, K. The use of
autologous platelet-rich plasma in the treatment of intractable skin ulcer: A case series. Open
J. Regen. Med. レフェリー有, 1, 2012, 29-32.
6. Kakudo, N., Kushida, S., Suzuki, K., Ogura, T., Notodihardjo, P.V., Hara, T. and Kusumoto,K. Effects of transforming growth factor-beta1 on cell motility, collagen gel contraction,
myofibroblastic differentiation, and extracellular matrix expression of human adipose-derived
stem cell. Human cell, レフェリー有, 25, 2012, 87-95.
7. 楠本健司, 福田智, 三宅ヨシカズ, 覚道奈津子, 櫛田哲史 多血小板血漿(PRP)と褥瘡、
難治性皮膚潰瘍の治療．日本医事新報, レフェリー無, 4626, 2012, 65-68.
8. Kushida, S., Kakudo, N., Suzuki, K. and Kusumoto, K. Effects of Platelet-Rich Plasma on
Proliferation and Myofibroblastic Differentiation in Human Dermal Fibroblasts. Ann. plast.
surg. レフェリー有, in press, 2012.
Books
1. Kakudo, N., Kushida, S., Ogura, T., Suzuki, K. and Kusumoto, K. Adipose-Derived StemCells and their Application in Tissue Engineering. Ed by Takeshi Ishikawa and Masayoshi
Yamamoto. Tissue Engineering: Fundamentals, Techniques and Applications, 5, 2012, 89-96.

2. Kakudo, N., Kushida, S., Saito, N., Suzuki, K., Kusumoto, K. Evaluation of the Effect of
Glycolic Acid Chemical Peeling on Facial Skin by Computer Analysis of Digital
Camera-Captured Images. Ed by Leon V. Berhardt Advances in Medicine and Biology, Nova
Science Publishers, 56, 2012, 149-154.
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3. Kakudo, N., Kushida, S., Saito, N., Suzuki, K., Kusumoto, K. A Procedure for the Assessmentof Skin Aging. Ed by Ayaka Ekiguchi and Moriko Imada Skin Aging: New Research, 13,2013, 131-136.
Proceedings
1. Kusumoto, K., Suzuki, K., Fukuda, S., Kakudo, N., Tanaka, Y., Sasao, S., Miyake, Y.,
Kushida, S., Priscilla, V. and Ogura, T. Autologous platelet-rich plasma(PRP) and theapplication for various wounds and poor conditions. The 11th Japan-Korea Congress of
Plastic and Reconstructive Surgery, Awaji. 2012.5.

2. Morimoto, N., Ito, T., Kanda, N., Kawai, K., Nakamura, Y., Teramukai, S., Yokode, M.,
Maekawa, T., Shimizu, A. and Suzuki, S. An exploratory clinical study on the safety andefficacy of an autologous fibroblast-seeded artificial skin cultured with animal product-free
medium in patients with diabetic foot ulcers. World Union of Wound Healing Societies2012.
Yokohama, 2012.9.
3. Kushida, S., Kakudo, N., Ogura, T., Suzuki, K. and Kusumoto, K. Effects of Platelet-Rich
Plasma on proliferation and myofibroblastic differentiation in human dermal fibroblasts.
World Union of Wound Healing Societies2012. Yokohama, 2012.9.
4. Ogura, T., Kakudo, N., Kushida, S., Suzuki, K. and Kusumoto, K. Platelet-Rich Plasma
promotes healing of mouse skin wounds and increases vascularization World Union of Wound
Healing Societies2012. Yokohama, 2012.9.
5. Notodihardjo, P.V., Kakudo, N., Suzuki, K., Yasui, M., Tabata, Y. and Kusumoto, K. A
combination of gelatin hydrogel and Platelet-Rich Plasma promotes wound healing in mice.
World Union of Wound Healing Societies2012. Yokohama, 2012.9.
6. Kusumoto, K., Suzuki, K., Fukuda, S., Kakudo, N., Tanaka, Y., Sasao, T., Miyake, Y.,
Kushida, S., Notodihardjo, P.V. and Ogura, T. Autologous Platelet-Rich Plasma(PRP) and theapplication for chronic wounds. World Union of Wound Healing Societies2012. Yokohama,2012.9.
7. Kusumoto, K. Autologous Platelet-Rich Plasma on wound healing and the practicalapplications. World Union of Wound Healing Societies2012. Yokohama, 2012.9.
8. Miyake, Y., Fukuda, S. and Kusumoto, K. The treatment with PRP(Platelet-Rich Plasma) forpressure ulcer pocket. World Union of Wound Healing Societies2012. Yokohama, 2012.9.
9. Fukuda, S., Nakano, M., Kusumoto, K. Wound healing-promoting effects of sulfadiazine
silver(GEBEN cream)-review of our series- World Union of Wound Healing Societies2012.
Yokohama, 2012.9.
10. Ogura, T., Kakudo, N., Kushida, S., Suzuki, K. and Kusumoto, K. Proliferative effect onhuman dermal fibroblasts using Platelet-Rich Plasma(PRP) prepared by an automatic blood
separation device "MAGELLAN" World Union of Wound Healing Societies2012. Yokohama,2012.9.
11. Kuro, A., Suzuki, K., Horio, O., Kakudo, N., Miyake, R., Toki, H., Kushida, S., Ogura, T. andKusumoto, K. Usefulness of voltage-applied unfrozen storage of blood vessels. World Union
of Wound Healing Societies2012. Yokohama, 2012.9.
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12. Morimoto, N., Rino, A., Fujitaka, J., Ito, R., Nakamura, Y., Kawai, K. and Suzuki, S.
Evaluation of neovascularization in a grafted bilayered artificial dermis and a cultured dermis
using laser doppler imaging World Union of Wound Healing Societies2012. Yokohama,2012.9.
13. Nakano, M., Fukuda, S., Miyake, Y., Kusumoto, K. Cystic tumors treatment with
Platelet-Rich Plasma(PRP) World Union of Wound Healing Societies2012. Yokohama,2012.9.
14. Kusumoto, K. New approaches for the use of PRP in chronic wounds. The 4th International
Regenerative Surgery. Rome, Italy, 2012.12.

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Annual Report 研究成果報告書
Title of Project

Cellular function imaging by x-ray microscopy
軟Ｘ線顕微鏡による細胞内機能イメージング

P.I.

Kuniko Kusumoto, 楠本(竹本)邦子

E-mailtakemoto@hirakata.kmu.ac.jp

Affiliation

Physics, 物理学講座

Collaborators

楠本（竹本）邦子、新庄正路、松村義隆、市村薫

Keywords

soft X-ray microscope, -CT、carboxysomepolyphosphate granule, XX-ray
solution scattering, folding

1. SUMMARY OF RESEARCH RESULTS
1. Soft X-ray microscopy imaging(1) Soft X-ray microscopic examinations of prokaryotic organelle
Recently, many biologists categorize any compartment bounded by a biological membrane with adedicated biochemical function as an organelle. Therefore a number of unique and diverse
organelles can be found in the prokaryotic. Unstained and unsliced air-dried cells of the
cyanobacterium, Phormidium tenue, were observed using soft X-ray microscope and some X-ray
absorbed structures were found in the cells. To identify the structures, the transmission electron
microscope (TEM) and low temperature / low vacuum scanning electron microscope (lowtemperature / low vacuum SEM) were done. Results of the observations showed that the structures
were carboxysome and polyphosphate granule.
(2) Soft x-ray micro computed tomography (-CT) observation of phytoplankton
We used soft X-ray tomography to study the 3D ultrastructural organization of whole cells of theunicellular green alga producing musty odor, Phormidium tenue.
P. tenue cells were encapsulated in a glass capillary tube and air-dried. 2.0 nm wavelength wasused for imaging. Forty five images were acquired with a rotation step of 4° and an exposure
time of 180 s. Cross sectional images by the CT were reconstructed by the convolution
back-projection method with Shepp-Logan filter. Air-dried cells were imaged using the natural
X-ray absorption contrast of them and it succeeded in acquisition of the first CT image.
2. Structural change of SH3 domain protein
We investigate structural changes of hNck2 SH3 domain protein.
This protein takes -structure rich conformation at native condition. It is known that hNck2 SH3domain takes equilibrium -helix-rich intermediate at acidic pH.
By X-ray solution scattering and structural calculation, we found the protein forms ellipsoidal
dimer in native condition and forms elongated monomer in acidic pH. Calculations aboutmolecular weight and pair distribution functions also support these results.
1. 軟 X 線顕微鏡による細胞内機能イメージング①原核細胞オルガネラの X 線顕微イメージング
シアノバクテリア中に存在する原核細胞オルガネラの X 線顕微イメージングを目指した。染色や薄片化していない微小シアノバクテリアを軟 X 線顕微鏡で観察し、細胞内部で顆粒状の構造体を可視することができた。透過型電子顕微鏡および走査型電子顕微鏡による観察を行い、これらの構造体はカルボキシソーム様構造体とポリリン酸顆粒であることを明らかにした。
②軟 X 線-CT 法によるシアノバクテリアの 3 次元微細構造観察
軟 X 線顕微鏡に 3 次元観察を導入し、X 線-コンピュータトモグラフィー(-CT)で細胞の 3 次元観察することを目指した。Phormidium tenue と同定されているシアノバクテリアを直径 10  m のガラスキャピラリィに封入し，結像型軟 X 線顕微鏡を用いて CT による-184-

3 次元観察した。回転角 4 度毎に 45 枚の投影像を取得し、断層像の再構成は Filtered
Backprojection 法で，Shepp-Logan フィルターを用いて行い、風乾状態の P. tenue の X 線 -CT
像を得ることができた。
2.SH3 タンパク質のとり得る構造多形
hNcK2 SH3 ドメイン蛋白について、構造の pH 依存性、濃度依存性を調べ酸性 pH で非天然構造モノマーとして存在し、中性ではダイマーに構造が変化する事を見出した。X 線の溶液散乱を行い散乱曲線から構造を計算し、中性 pH では確かに体積の大きなダイマーを形成している事、その形状は楕円体的である事等が分かった。又、酸性 pH では天然構
造は壊れているが、非天然の-helix を含んで広がった構造をとっていてランダムコイル
では無い状態であると理解される。
優れた成果があがった点
シアノバクテリアを丸ごと観察し、原核細胞オルガネラの存在を確認と X 線 -CT 像の取得に成功した。
X 線溶液散乱の結果から分子構造、体積、分子量を検討してこの蛋白質が酸性で α-helixに富むモノマー、中性 pH でダイマーをとる事、中性でも希薄溶液下ではモノマーとなる事を明らかにした。
問題点
軟 X 線顕微鏡による細胞内機能イメージングにおいては、含水状態で P. tenue の X 線-CT 撮影も試みたが、時間経過と共に試料が動き、3 次元像を得るに至らなかった。次年
度は、含水状態での試料固定法を検討し，含水状態での X 線-CT 像の獲得を目指す。

2. List of Publications
Original Articles
1. Matsumura, Y., Shinjo, M., Matsui, T., Ichimura, K., Song, J. and Kihara H. Structural studyof hNck2 SH3 domain protein in solution by circular dichroism and X-ray solution scattering.
Biophys. Chem. レフェリー有, 175-176, 2013, 39-46.

2. 竹本邦子, 木原裕 他 5 名 琵琶湖産の糸状カビ臭産生藍藻 Phormidium tenue の細胞内微細構造観察：軟 X 線顕微鏡と透過型電子顕微鏡および低真空クライオ走査型電子顕微鏡を用いた比較観察 日本水処理生物学会誌, レフェリー有, 48, 2012, 157-163.
3. Takemoto, K., Ikegaya, H., Yaji, T., Ichise, S., Ohta, T. and Kihara, H. Infrared
Micro-Spectroscopy Observation of plantplankton with in Lake Biwa. Memoirs of the SR
Center, Ritsumeikan University, レフェリー有, 14, 2012, 173-17
4. Takemoto, K., Kihara, H. et al.Soft X-ray Imaging of Intracellular Structure of Leptolyngbya
tenuis (Phormidium tenue) generating musty smell in Lake Biwa. Memoirs of the SR Center,
Ritsumeikan University, レフェリー有, 14, 2012, 171-172.
5. Matsumura, Y., Matsui, T., Ichimura, K., Song, J. and Kihara, H. Structural study of hNck2
SH3 domain protein by X-ray solution scattering I. Concentration dependence at pH 6. Photon
Factory Activity Report, レフェリー無, 29, 2012, 368.
6. Matsumura, Y., Shinjo, M., Matsui, T., Ichimura, K., Song, J. and Kihara, H. Structural studyof hNck2 SH3 domain protein by X-ray solution scattering II. pH-dependent structuralchange. Photon Factory Activity Report, レフェリー無, 29, 2012, 367.
7. Matsumura, Y., Shinjo, M., Matsui, T., Ichimura, K., Song, J. and Kihara, H. Structural studyof hNck2 SH3 domain protein by X-ray solution scattering III. Structure of hNck2 SH3domain at pH2. Photon Factory Activity Report, レフェリー無, 29, 2012, 365.
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8. Shinjo, M., Matsumura, Y., Ichimura, K., Song, J. and Kihara, H. Structural study of hNck2
SH3 domain protein by X-ray solution scattering IV. Structural analysis of hNck2 SH3 domain
at pH 2 and pH 6: non-native alpha-helix-rich monomer and native dimer. Photon Factory
Activity Report, レフェリー無, 29, 2012, 366.
Proceedings
1. 竹本邦子, 木原 裕 他 4 名. 結像型軟 X 線顕微鏡試料システム. 第 26 回日本放射光学会年会・放射光科学合同シンポジウム, 名古屋市, 2013.1.

2. 吉村真史, 竹本邦子, 大東琢治, 木原裕 他 2 名 軟 X 線顕微鏡による軽元素マッピ
ング. 第 26 回日本放射光学会・放射光科学合同シンポジウム, 名古屋市, 2013.1.
3. Matsumura, Y., Shinjo, M., Matsui, T., Ichimura, K., Liu, J., Song, J. and Kihara, H.
Equilibrium expanded non-native alpha-helix intermediate of hNck2 SH3 domain at acidic
pH. The 6th International Symposium “Molecular Science of Fluctuations toward Biological
Functions”, 京都, 2012.12.
4. Matsumura, Y., Jin, X., Shinjo, M., Kimura, S. and Kihara, H. Teraherz spectroscopy ofproteins by synchrotron radiation at UVSOR. The 6th International Symposium “Molecular
Science of Fluctuations toward Biological Functions”, 京都, 2012.12.
5. Shinjo, M., Matsumura, Y., Ichimura, K., Song, J., Kihara, H. Structural analysis of hNck2
SH3 domain at various pH: non-native -helix-rich monomer and native dimer. 第 50 回日本
生物物理学会年会, 名古屋, 2012.9.
6. Matsumura, Y., Shinjo, M., Matsui, T., Ichimura, K., Song, J., Kihara, H. Structural properties
and folding process of hNck2 SH3 domain.第 50 回日本生物物理学会年会, 名古屋, 2012.9.
7. Takemoto, K., Mizuta, G., Yamamoto, A., Yoshimura, M., Ichise, S., Namba, H. and Kihara,
H. Soft X-ray Imaging of Intracellular Granules in Filamentous Cyanobacterium. 11th
International Conference on X-ray Microscopy, Shanghai, China, 2012.8.
8. Takemoto, K., Kishin, U., Ohigashi, T., Fujii, H., Yoshimura, M., Namba, H. and Kihara, H.
Improvement of Cryogenic 3-Dimensional Observation System of Soft X-ray Microscope at
the SR Center of Ritsumeikan University. 11th International Conference on X-ray Microscopy,
Shanghai, China, 2012.8.
9. 松村義隆, 新庄正路, 木原裕. 他 3 名 X 線溶液散乱による hNck2 SH3 domain 蛋白質の構造研究 その 1. 第 21 回日本バイオイメージング学会学術集会, 京都, 2012.8.
10. 松村義隆, 新庄正路, 木原裕. 他 3 名 X 線溶液散乱による hNck2 SH3 domain 蛋白質の構造研究 その 2. 第 21 回日本バイオイメージング学会学術集会, 京都, 2012.8.
11. 新庄正路, 松村義隆, 木原裕. 他 4 名 src SH3 ドメインにおける初期フォールディング
中間体の構造解析 II -Local Minimum 構造の探索-. 第 21 回日本バイオイメージング学会学術集会, 京都, 2012.8.
12. 竹本邦子, 木原裕. 他 4 名 軟 X 線顕微鏡によるピコ植物プランクトンの粘質鞘の解析. 第 21 回日本バイオイメージング学会学術集会, 京都, 2012.8.
13. 竹本邦子, 木原裕 他 5 名. 軟 X 線顕微鏡による糸状植物プランクトンの細胞内顆粒の観察. 第 21 回日本バイオイメージング学会学術集会, 京都, 2012.8.
14. 木原裕, 竹本邦子, 他 4 名 生きた細胞の動きを観測する軟Ｘ線顕微鏡の開発. 第 21 回
日本バイオイメージング学会学術集会, 京都, 2012.8.
15. Matsumura, Y., Shinjo, M., Ichimura, K., Matsui, T., Song, J. & Kihara, H. Structural featuresof hNck2 SH3 domain, (1P-018, 6/20). 第 12 回日本蛋白質科学会年会, 名古屋, 2012.6.
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私立大学戦略的研究基盤形成支援事業（平成 23 年～27 年）
MEXT– supported Program for the Strategic Research
Foundation at Private Universities (2011－2015)

分子イメージングによる体系的病態の解明と診断治療法の開発
Systematic elucidation of pathogenesis anddevelopment of diagnosis and treatment bymolecular imaging
平成 23-24 年度研究成果中間報告書
Annual Report 2011-12
関西医科大学
Kansai Medical University
平成 25 年 9 月
September 2013-188-