Niigata Dent. J. 43（2）：17 － 24,

2013

105

－原著－
口腔粘膜上皮細胞培養上清で培養した口腔粘膜線維芽細胞の形質に関する検討
マザビンビンタモニル
新潟大学大学院医歯学総合研究科

口腔解剖学分野

Characterization of an oral fibroblast phenotype cultured in oral keratinocyteconditioned medium
Mah Zabin Binta Monir
Division of Oral Anatomy, Department of Oral Biological Science, Niigata University Graduate School of Medical and Dental Sciences
平成 25 年９月 17 日受付

平成 25 年９月 20 日受理

キーワード：口腔粘膜線維芽細胞，口腔粘膜角化細胞，培養上清，形質，単層培養
Key words: Oral fibroblast, oral keratinocyte, conditioned medium, phenotype, monolayer culture
Abstract:
Fibroblasts in monoculture grown in serum-containing medium are highly-proliferating. However, datasuggested that use of static fibroblasts provides a better model to study biological phenomena than proliferating
fibroblasts. Since previous studies have stated keratinocyte-conditioned medium reduced fibroblast proliferation,
this study aimed to examine if the oral keratinocyte (OK) -conditioned medium (CM) can decrease oral fibroblasts(OF) proliferation and to characterize their phenotype. Primary human OK and OF were grown in a completed
EpiLife®（0.06mM Ca++）and Dulbecco's modified Eagle medium (DMEM) containing 10% calf serum (DMEM-CS),
respectively. OK-CM was conditioned for 24 hours in a near-confluent OK culture. OFs plated in a micro-plate well
were cultured with DMEM-CS, serum-free DMEM (SF-DMEM) and OK-CM for up to 96 hours. Proliferation rateand cell cycle profile were analyzed using a MTT assay and a fluorescence-activated cell sorter. The "phenotypicchanges" of OFs were determined by the activity of senescent-associated β-galactosidase (β-gal) and the secreted
protein levels including keratinocyte growth factor (KGF), human type I collagen and matrix metalloproteinase-1(MMP-1) measured by enzyme-linked immunosorbent assay (ELISA). The proliferating rate and the proportion ofcells in S phase were significantly lower when cells were cultured in OK-CM. The β-gal activity suggested OFsin OK-CM still had proliferating potential. ELISA assay showed OFs cultured in OK-CM produce KGF and MMP-1
as did OFs grown in DMEM-CS while their ability to produce type I collagen was significantly lower than OFs inDMEM-CS. This study suggested the OK-CM generated a quiescent OF population possessing the characteristicof extracellular matrix degradation rather than synthesis.
MTT : 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
日本語要旨：
生体反応の研究に用いる線維芽細胞は増殖している細胞ではなく休止期のものが好ましい。上皮細胞の培養上清に線維芽細胞の増殖抑制効果があることが報告されている。本研究の目的は口腔粘膜上皮細胞の培養上清が培養口腔粘膜線維芽細胞の増殖性を抑え，細胞形質を変化させるかを検証することである。ヒト初代口腔粘膜上皮細胞（OK）と線維芽細胞（OF）はそれぞれ EpiLife と 10％ウシ胎児血清含有ダルベッコ改変イーグル培地（DMEM）（DMEM-CS）中で培養した。コンフルエントに近い口腔粘膜上皮細胞を 24 時間培養したものを上清とした。口腔粘膜線維芽細胞
は DMEM-CS，DMEM 培地単独（SF-DMEM），口腔粘膜上皮細胞培養上清（OK-CM）の３種類の培地で，最長 96－ 17 －106

新潟歯学会誌43（2）：2013

時間培養した。細胞増殖能と細胞周期はそれぞれ MTT assay とセルソーターで解析した。細胞形質変化の分析はさらに，βガラクトシダーゼ活性発現とエライザ法（ELISA）による各種タンパク質（ケラチノサイト増殖因子（KGF），タイプ I コラーゲン，マトリックスメタロプロテアーゼ -1（MMP-1））分泌量で検討した。細胞増殖能と S 期の細胞の割合は有意に OK-CM で培養した細胞で低下しており，G0/G1 期停止による細胞増殖抑制の可能性が示唆された。
また OK-CM 培養では，βガラクトシダーゼ活性を示した細胞は少数で，口腔粘膜線維芽細胞は休止期にいるが，増殖能が失われていないことを示した。KGF と MMP-1 の産生量は，DMEM-CS で培養した細胞と同等であったのに対し，I 型コラーゲンの産生量が有意に低かった。本研究から，OK-CM は培養口腔粘膜線維芽細胞の増殖能を抑え，かつ細胞外基質分解性の細胞形質となることが示唆された。

decreased10.11）. Those fibroblast phenotypic changes

Introduction

were confirmed to be beneficial to dermal tissue
regeneration12）. Although most products commerciallyConstant tissue renewal of stratified squamous

available such as Dermagraft®and Apligraf®

epithelia such as skin and oral mucosa is maintained

incorporate normal proliferating early passagedby a balance between cell differentiation andfibroblasts from foreskin to fabricate the tissue-desquamation at the epithelial surface and theengineered substitutes, there is a body of data

replenishment of keratinocytes by cell divisionsuggesting that use of static fibroblasts is a betteroccurred in basal and parabasal cell layers1）. Thus,

model to address biological phenomena than primary

mitotic activity of keratinocytes is frequently

proliferating fibroblasts even when fabricating 3D

recognizable in vivo. In contrast to keratinocytes,

tissue-engineered constructs5）. Thus, to implement a

fibroblasts in normal adult skin and oral mucosa

better model, we should use different culture techniqueinfrequently divide and are static in nature2.3）. In

addition, in an uninjured tissue, they are relatively

that allows to provide quiescent fibroblasts even in amonolayer culture system.

inactive in terms of protein synthesis and thin in

There have been two means to modulate fibroblast

histological appearance4）. However, the wound healing

phenotype to a static and non-proliferating natureprocess induces phenotypic alterations in the resident

mimicking residential fibroblasts in dermal tissue. One

fibroblast population from quiescence to migration andis utilization of irradiated dermal fibroblasts13）, and theproliferation, contracting cells and producing

other is use of keratinocyte-conditioned medium

e x t r a c e l l u l a r m a t r i x 5）. S i n c e f i b r o b l a s t s i n a(CM)14）. Previous reports stated skin keratinocyte-CM

monoculture are invariably highly proliferative in a

reduced skin fibroblast proliferation as well as collagenserum-containing medium, this fibroblast phenotype

matrix synthesis2.15,16）. In the oral mucosa, interactions

seems to be "activated" fibroblasts in vivo in response

between keratinocyte and fibroblast are also critical tot o w o u n d h e a li ng4）

. W hil e st udi e s h av e u s e dbasic research as a tool to translate into regenerative

proliferating fibroblasts in a monoculture to examine

medicine. However, there have been few studies on

the cytotoxic effects of pharmaceuticals and chemicals

their cross-talk by using oral keratinocytes-conditioned

and consequent cellular responses6.7）, data obtained

medium (OK-CM)17）. Consequently, we are interested

from those experiments cannot be extrapolated to thein the feasibility of OK-CM that is always discarded ineffects in the target tissue in vivo because their

our laboratory.

cellular responses may be different from those of

In this study, we hypothesized the OK-CM yielded

inactive fibroblasts, similar to the phenotype in an

in our laboratory might contribute to oral fibroblast

uninjured tissue.

(OF) phenotypic changes from an actively-cycling state

Recently, researchers have paid an attention toto a more static state. Thus, this study aimed totissue engineering as an emerging technology for

examine if the OK-CM can decrease OFs proliferation

regenerative medicine. In contrast to a monolayer

in a monolayer culture. In addition, we further

culture system, it is well-known that cells cultured in acharacterized the phenotypic changes of OFs cultured

three-dimensional (3D) scaffold behave differently

8.9）

. In

in OK-CM. The re-use of OK-CM could provide anfact, the proliferation rate and collagen production byeasier and inexpensive approach for studying thefibroblasts embedded in a 3D collagen matrix

cellular biology of OFs.

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