近畿大学次世代基盤技術研究所報告 Vol. 5（2014）49－53

8&67ᆺࢫࣝ࣍࣋ࢱ࢖ࣥ⣔࣏࣐࣮ࣜࢆ⾲㠃ಟ㣭ࡋࡓ࢞ࣛࢫᇶᯈࡢㄪ〇࡜+H/D⣽⬊ࡢᗘ่⃭ࡣࡃ㞳Preparation of UCST types of thermoresponsive sulfobetaine polymer modified glass andthermal stimuli-exfoliation of HeLa cells on the substrate        ◊✲௦⾲⪅ ኱Ꮫ㝔ࢩࢫࢸ࣒ᕤᏛ◊✲⛉*1   ᩍᤵ ⓑ▼ ᾈᖹ                 Kohei Shiraishi
ᩍᤵ ᒣ⏣ ᗣᯞ         ඹྠ◊✲⪅ ኱Ꮫ㝔ࢩࢫࢸ࣒ᕤᏛ◊✲⛉*1
Yasue Yamada኱Ꮫ㝔ࢩࢫࢸ࣒ᕤᏛ◊✲⛉*1 ኱Ꮫ㝔⏕ ௒ᇛ ᫂඾Akinori Imajo኱Ꮫ㝔ࢩࢫࢸ࣒ᕤᏛ◊✲⛉*1 ኱Ꮫ㝔⏕ ఀ⸨ ኱᫬Taiji Ito኱Ꮫ㝔ࢩࢫࢸ࣒ᕤᏛ◊✲⛉*1 ኱Ꮫ㝔⏕ ㎰᐀ ㎮ᕫTatsuki Nousou

Poly { 2-[ (Methacryloyloxy) ethyl ] dimethyl - (3-sulfoproryl) ammonium hydroxide } [ poly(SBMA)] with upper critical solution temperature(UCST) was grafted onto a glass substrate bysurface initiated radical polymerization. From the scanning probe microscopy(SPM)
measurements, poly(SBMA) brushes were observed on the glass substrate(g-glass). From thecontact angle(ǉ) measurements to water at 26Υ, the ǉ values of g-glass increased from
ǉ=21°(the untreated glass) to ǉ=54°. On the other hand, the ǉ values at 40Υ, the ǉ values ofg-glass decreased from ǉ=54° to ǉ=45°. After being HeLa cells attached and proliferated on theg-glass at 30Υ under UCST of poly(SBMA) burshes, The HeLa cells attached were thermallyexfoliated by increasing culture temperature over 37Υ for 2 h. The number of thermal
exfoliation of HeLa cells on the g-glass was higher than that on untreated glass.
Keywords: 2-[(Methacryloyloxy)ethyl]dimethyl-(3-sulfoproryl)ammonium hydroxide,
Upper critical solution temperature (UCST), Surface initiated polymerization,
Thermal exfoliation, HeLa cells㸬ࡣࡌࡵ࡟ ᖿ⣽⬊ࢆ⏝࠸ࡿᕤᏛศ㔝ࡢ◊✲㛤Ⓨࡀ๰⸆ࢫࢡ࣮ࣜࢽࣥࢢࡸ෌⏕་⒪࡬ࡢᛂ⏝ࡢほⅬ࠿ࡽάⓎ໬ࡋ࡚࠸ࡿ㸬࡜ࡃ࡟㸪ேᕤከ⬟ᛶᖿ⣽⬊ (iPS ⣽⬊: induced pluripotentstem cells)(1)ࡢస〇ᢏ⾡ࡢ☜❧ࡣ㸪ᝈ⪅⮬㌟ࡢ iPS ⣽⬊࠿ࡽศ໬ㄏᑟࡋࡓ⣽⬊ࢆά⏝ࡋ࡚㸪࣮࢜ࢲ࣮࣓࢖ࢻ๰⸆ཬࡧᣄ⤯཯ᛂࡢ࡞࠸⮚ჾస〇➼ࡀ⌧ᐇ໬ࡋ࡚࠸ࡿ(2),(3)㸬ࡋ࠿ࡋ iPS ⣽⬊➼㸪ᖿ⣽⬊ࡢᐇ⏝໬࡟ࡣ㸪ձiPS ⣽⬊స〇ࡢ㧗ຠ⋡໬㸪ղ⣽⬊ᶵ⬟ࡢ⢭ᐦデ᩿㸪ճiPS ⣽⬊స〇᫬ࡲࡓࡣ⣽⬊ศ໬ㄏᑟ᫬ࡢ୙⏝⣽⬊ࡢ㝖ཤ㸪մ┠ⓗ⣽⬊ࡢ㑅ᢥⓗ࣭㠀౵くⓗ࡞ᅇ཰➼ࡢㄢ㢟ࡶᑡ࡞ࡃ࡞࠸㸬ㄢ㢟ゎỴࡢࡓࡵ࡟ࡣ㸪⣽⬊᧯స࡟ಀࡿ⣲ᮦࡸ࿘㎶ᶵჾ࠶ࡿ࠸ࡣჾᲔࡶᚲせ࡛㸪ࣟ࣎ࢸ࢕ࢡࢫᢏ⾡ࢆే⏝ࡋࡓ⮬ື໬ࢩࢫࢸ࣒ࢆ⏝࠸ࡓデ᩿ཬࡧᅇ཰ࡢࣁ࢖ࢫ࣮ࣝࣉࢵࢺ໬࣭పࢥࢫࢺ໬࣭⿦⨨ᑠᆺ໬➼ࡢᚲせࡀ࠶ࡿ㸬⌧ᅾ㸪⣽⬊デ᩿ࢆከ᳨యྠ᫬࡟㸪࠿ࡘࣁ࢖ࢫ࣮ࣝࣉࢵࢺ࡟⾜࡞࠼ࡿࢶ࣮ࣝ࡜ࡋ࡚⣽⬊࣐࢖ࢡࣟ࢔ࣞ࢖(ǍAy)ࡀὀ┠ࡉࢀ࡚࠸ࡿ(4),(5)㸬≉࡟㸪ǍAy ୖ࡬⣽⬊ࢆ➼㛫㝸࡟㓄⨨ࡍࡿᡭἲࡣ㸪ᶵ⬟ゎᯒࡢࡓࡵࡢࢶ࣮ࣝ࡜ࡋ࡚ὀ┠ࡉࢀ࡚࠾ࡾ㸪๰⸆ࡢࡓࡵࡢ⸆๣ࢫࢡ࣮ࣜࢽࣥࢢ(6)~(8)㸪་⸆ရࡸ໬Ꮫ≀㉁࡞࡝ࡢ᭷⏝ᛶࡸᏳ඲ᛶࡢホ౯(9)~(11)㸪⣽⬊ࡢศ໬ㄏᑟᅉᏊࡸ㑇ఏᏊ⩌ࡢࡲࡓ㸪ࢫࢡ࣮ࣜࢽࣥࢢ(12)~(14)࡞࡝ǍAyࡢྍ⬟ᛶࡣ኱ࡁ࠸㸬⣽⬊ᶵ⬟ࡢデ᩿࡛ࡣ㸪ᚑ᮶ࡲ࡛ࡣከࡃࡢ⣽⬊࠿ࡽ୍ᣓ࡛ᚓ࡚࠸ࡓ᝟ሗࢆ㸪୍ಶ࠶ࡿ࠸ࡣᩘಶࡢ㝈ࡽࢀࡓ⣽⬊ẖ࡟ᚓࡽࢀࡿ㸬ࡋࡓࡀࡗ࡚⣽⬊᥋╔㠃࡜㠀᥋╔㠃ࢆᐃᗎⓗ࡟㓄ิࡋࡓ ǍAy ࡣ⣽⬊ࡢ⢭ᐦ࡞デ᩿ࢶ࣮ࣝ࡜ࡋ࡚ࡢᶵ⬟ࢆ᭷ࡋ࡚࠸ࡿ࡜⪃࠼ࡽࢀࡿ㸬ⴭ⪅ࡽࡣ㸪พ㒊ࢆ⣽⬊᥋╔㠃ࡢ࢞ࣛࢫࢫ࣏ࢵࢺ㸪ฝ㒊ࢆ⣽⬊㠀᥋╔㠃ࡢ㔠⣲ᮦ࡜ࡋࡓ*ཎ✏ཷ௜ 2014 ᖺ 5 ᭶ 12 ᪥
*1 ࠛ739-2116 ᮾᗈᓥᕷ㧗ᒇ࠺ࡵࡢ㎶ 1 ␒  E-Mail siraisi@hiro.kindai.ac.jp－ 49 －

ǍAy ࢆ⏝࠸࡚㸪ಶ⣽⬊ࡢデ᩿ཬࡧ㠀౵くⓗ࠿ࡘ㑅ᢥⓗ࡟ᅇ཰ࡋᚓࡿᢏ⾡ࡢ㛤Ⓨࢆ⾜࡞ࡗ࡚࠸ࡿ(15),(16)㸬ࡇࡢ࡜ࡁ㠀౵くⓗ࡞⣽⬊ᅇ཰ᶵ⬟ࢆ୚࠼ࡿࡓࡵ㸪 ᗘᛂ⟅ᛶ࣏࣐࣮ࣜࡢᅛᐃ໬ࢆ᳨ウࡋ࡚࠸ࡿ㸬ୗ㝈⮫⏺ඹ⁐ ᗘ(LCST)ᆺ
ᗘᛂ⟅ᛶ࣏࣐࣮ࣜࡣ㸪⁐ᾮ୰ࡢ ᗘኚ໬࡛ぶ࣭␯Ỉᛶࡀ㸪┦㌿⛣ ᗘࢆቃ࡟ప 㸦ぶỈᛶ㸧я㧗 㸦␯Ỉᛶ㸧࡜ྍ㏫ⓗ࡟ኚ໬ࡍࡿ㸬LCST ᆺ࣏࣐࣮ࣜࢆ⾲㠃࡟ᅛᐃ໬ࡋࡓ⣲ᮦࡣ㸪᥋╔⣽⬊ࡢᇵ㣴 ᗘࢆపୗࡉࡏ㠀౵くⓗ࡟⣽⬊ᅇ཰ࡍࡿ᪉ἲ࡜ࡋ࡚ᐇ⏝໬ࡉࢀ࡚࠾ࡾ(17),(18)㸪ǍAy ࢆ⏝࠸ࡓ⣽⬊ࡢ㑅ᢥⓗᅇ཰࡬ࡢᛂ⏝ࢆ㐍ࡵ࡚࠸ࡿ㸬୍᪉㸪
LCST ᆺ࣏࣐࣮ࣜᅛᐃ໬ ǍAy ୖ࡛ࡢ㑅ᢥⓗ࡞ᅇ཰࡛ࡣ㸪⿦⨨ࡀ」㞧࡜࡞ࡾ ǍAy ࢫ࣏ࢵࢺࡢᒁᡤࢆ෭༷ࡍࡿ࠿㸪࣮ࣞࢨ࣮ග࡛୙⏝⣽⬊ࢆணࡵ㝖ཤࡋࡓࡢࡕ㸪ǍAy ඲యࢆ෭༷ࡋ࡚┠ⓗ⣽⬊ࢆᅇ཰ࡍࡿ᪉ἲࢆ㛤Ⓨࡋ࡚࠸ࡿ(17)㸬ᮏǍAy ࡛ࡣ┠ⓗ⣽⬊ࡀᴟᑡᩘࡢሙྜ࡟ᛂ⏝ࡀࡋ࡟ࡃ࠸ࡇ࡜࠿ࡽ㸪LCST ᆺ࣏࣐࣮ࣜ࡜㏫ࡢ㧗 ࡛ぶỈᛶࢆ♧ࡋప࡛␯Ỉᛶࢆ♧ࡍୖ㝈⮫⏺ඹ⁐ ᗘ(UCST)ᆺ ᗘᛂ⟅ᛶ࣏࣐࣮ࣜ(19),(20)ࢆ⏝࠸㸪ǍAy ࢫ࣏ࢵࢺ࡟ᅛᐃ໬ࡋ࡚㸪㧗⢭ᗘ࡞఩⨨Ỵࡵ CW ࣮ࣞࢨ࣮ග↷ᑕ࡟ࡼࡿᒁᡤຍ ࡼࡿ㑅ᢥⓗ࡞ᅇ཰ࡢᇶ♏ⓗ࡞▱ぢࢆᚓࡿࡓࡵ㸪ǍAy ࢫ࣏ࢵࢺ࡜ྠࡌ࢞ࣛࢫ㠃࡟ UCST ᆺ࣏࣐࣮ࣜࡢ⾲㠃㛤ጞࣛࢪ࢝ࣝ㔜ྜἲ࡟ࡼࡿᅛᐃ໬ࢆヨࡳࡓ㸬ࡇࡇ࡛ࡣ㸪⏕య㐺ྜᛶᮦᩱ࠶ࡿ࠸ࡣ⏕య≀㉁ࡢ᥋╔ᢚไຠᯝࡶ♧ࡍ⣲ᮦ(21)࡛᥋╔⣽⬊࡬ࡢࢲ࣓࣮ࢪࡢ㍍ῶ࡜᥋╔ᛶไᚚࡀᐜ᫆࡜⪃࠼ࡽࢀࡿ୧ ᛶ ࢖ ࢜ ࣥ ᵓ 㐀 Poly { 2-[(Methacryloyloxy) ethyl]
dimethyl-(3-sulfo- proryl)ammonium hydroxide}[poly(SBMA)] ࢆ UCST ࣏࣐࣮ࣜ (22) ࡜ࡋ࡚㑅ᢥࡋ㸪poly(SBMA)ᅛᐃ໬࢞ࣛࢫ(g-glass)ୖ HeLa ⣽⬊ࢆᇵ㣴࠾ࡼࡧຍ ࡟ࡼࡿࡣࡃ㞳ࢆᮍಟ㣭࢞ࣛࢫ➼࡜ẚ㍑᳨ウࡋࡓ㸬ᐇ㦂᪉ἲ ヨ⸆◲㓟㸦࿴ග⣧⸆ ᰴ 〇㸧㸪㐣㓟໬Ỉ⣲㸦࿴ග⣧⸆ ᰴ 〇㸧㸪3-࢔࣑ࣀࣉࣟࣆࣝࢺ࣓ࣜࢺ࢟ࢩࢩࣛࣥ㸦ᮾி໬ᡂ ᰴ 〇㸧㸪N,N-ࢪ࣓ࢳ࣒ࣝ࣍ࣝ࢔࣑ࢻ㸦⬺Ỉ㸧(DMF) 㸦࿴ග⣧⸆ᰴ 〇㸧㸪ࢺ࢚ࣜࢳࣝ࢔࣑ࣥ㸦࿴ග⣧⸆ ᰴ 〇㸧
㸪 2-࣓ࢱ
ࢡࣜࣟ࢖ࣝ࢜࢟ࢩ㸧࢚ࢳࣝࢪ࣓ࢳࣝ-3-ࢫࣝ࣍ࣉࣟࣆࣝ࢔
ࣥࣔࢽ࣒࢘ࣄࢻࣟ࢟ࢧ࢖ࢻ(SBMA) Sigma-ALDRICH〇 㸪4,4’-࢔ࢰࣅࢫ㸦4-ࢩ࢔ࣀྜྷⲡ㓟ࢡࣟࣛ࢖ࢻ㸧(V501-Cl)
ࡣ 4,4'-࢔ࢰࣅࢫ㸦4-ࢩ࢔ࣀྜྷⲡ㓟㸧(V501)㸦࿴ග⣧⸆ ᰴ〇㸧࡜஬ሷ໬ࣜࣥ(PCl5)㸦࢟ࢩࢲ໬Ꮫ ᰴ 〇㸧࠿ࡽྜᡂࡋ
ࡓ㸬࢚ࢱࣀ࣮ࣝ㸪ࢡ࣒ࣟࣟ࣍ࣝࡢ⁐፹ࡣ㸪ᖖἲ࡟ࡋࡓࡀࡗ࡚⢭〇ࡋࡓ㸬࣓ࢱࢡࣜࣝ㓟 n-ࣈࢳࣝ(BMA) 㸦࿴ග⣧⸆ ᰴ 〇㸧ࡣ[60Υ/30 mmHg]࡛ῶᅽ⵨␃ࡋ࡚⏝࠸ࡓ㸬⣧Ỉࡣ MILLIPORE 〇 MILL-Q Synthesis A10 ㉸⣧Ỉ〇㐀⿦⨨ࢆ⏝࠸࡚⢭〇ࡋ࡚౑⏝ࡋࡓ㸬࢝ࣂ࣮࢞ࣛࢫࡣ

Microscope Cover Glass(Ǘ=12 mm)㸦Fisher Scientific〇㸧ࢆࡑࡢࡲࡲ⏝࠸ࡓ㸬Minimum Essential Medium(MEM)㸦GIBCO 〇㸧㸪∵⫾ඣ⾑Ύ(FCS) 㸦GIBCO 〇㸧ࡣᖖἲ࡟ᚑࡗ࡚ㄪᩚࡋ࡚⏝࠸ࡓ㸬㠀ᚲ㡲࢔࣑ࣀ㓟(NEAA)
ࡣࡑࡢࡲࡲ⏝࠸ࡓ㸬ࣜࣥ㓟⦆⾪⏕⌮㣗ሷỈ(PBS)(pH7.4)
ࡣᖖἲ࡟ᚑࡗ࡚ㄪᩚࡋ࡚⏝࠸ࡓ㸬0.25% Trypsin ࡣᖖἲ࡟ᚑࡗ࡚ㄪᩚࡋ࡚⏝࠸ࡓ㸬⁛⳦Ỉࡣ⣧Ỉࢆ࣮࢜ࢺࢡ࣮ࣞࣈ⁛⳦ࡋ࡚⏝࠸ࡓ㸬3RO\ 6%0$ ࢢࣛࣇࢺ໬>SRO\ 6%0$ JJODVV@6%0$
%0$ ඹ 㔜 ྜ య ࢢ ࣛ ࣇ ࢺ ໬ ࢞ ࣛ ࢫ >SRO\ 6%0$FR
%0$ JJODVV@ࡢㄪ〇࢞ࣛࢫ〇ࢩ࣮ࣕࣞࢆ⏝࠸㸪ࣆࣛࣥࣁ⁐ᾮ>30㸣㐣㓟໬Ỉ
⣲/18 M ◲㓟=1/3(v/v)@2 mL ࡟࢝ࣂ࣮࢞ࣛࢫࢆධࢀ 1 hᾐₕࡋ㸪⣧Ỉ࡛࢝ࣂ࣮࢞ࣛࢫࢆὙίࡋࡓᚋ㸪࢚࢔࣮ࢥࣥࣉࣞࢵࢧ࣮࡛✵Ẽࢆ྿ࡁ௜ࡅ࡚஝⇱ࡋࡓ㸬Ὑίᚋ㸪0.179g (1 mM) 3-࢔࣑ࣀࣉࣟࣆࣝࢺ࣓ࣜࢺ࢟ࢩࢩࣛࣥࡢ DMF⁐ᾮ 1 mL ࡀධࡗࡓ࢞ࣛࢫ〇ࢫࢡ࣮ࣜࣗ⟶࡟࢝ࣂ࣮࢞ࣛ
ࢫࢆධࢀ㸪60Υ㸪3 h ཯ᛂࡋࡓ㸬ࡑࡢᚋ㸪DMF ࡛⾲㠃ࢆὙίࡋ㸪80Υ㸪4 h ஝⇱ჾ࡛↝ࡁ௜ࡅ࣭஝⇱ࢆ⾜ࡗࡓ㸬↝ࡁ௜ࡅᚋ㸪࢚ࢱࣀ࣮࡛ࣝ㉸㡢ἼὙί 10 min ⾜ࡗࡓ㸬࢞ࣛ ࢫ 〇 ࢫ ࢡ ࣜ ࣗ ࣮ ⟶ ࡟ ࢔ ࢰ 㛤 ጞ ๣ 0.0315 g (0.1mmol)V501-Cl ࢆ 1 mL ࡢࢡ࣒ࣟࣟ࣍ࣝ࡟⁐ゎࡋ㸪30.6
ǍL ࢺ࢚ࣜࢳࣝ࢔࣑ࣥࢆຍ࠼ࡓ㸬ࡑࡢΰྜࡋࡓࢡࣟࣟ࣍ࣝ
࣒⁐ᾮ࡟࢝ࣂ࣮࢞ࣛࢫࢆᾐࡋ㸪ᜏ ᵴ࡛ 30Υ㸪20 h ཯ᛂࡋࡓ㸬཯ᛂ⤊஢ᚋ㸪࣓ࢱࣀ࣮ࣝ࡟ᾐࡋ㸪ᐊ ࡛ 25 min㉸㡢ἼὙίࡋࡓ㸬࢞ࣛࢫ〇ࢫࢡ࣮ࣜࣗ⟶࡟ SBMA 0.264g(0.4725 M)㸪⣧Ỉ࡜࢚ࢱࣀ࣮ࣝࢆࡑࢀࡒࢀ 0.674 mL㸪
1.326 mL ࡢΰྜ⁐ᾮ 2 mL ࡟ㄪ〇ࡋࡓࡶࡢࢆຍ࠼㸪
V501-Cl ࢆᅛᐃ໬ࡋࡓ࢝ࣂ࣮࢞ࣛࢫࢆᾐࡋ㸪ᾮయ❅⣲ᾎ୰࡛෾⤖㸫⬺Ẽ㸫❅⣲⨨᥮ࢆ 3 ᅇ⧞ࡾ㏉ࡋࡓᚋ㸪80Υ࡛
20 h 㔜ྜࡋࡓ㸬཯ᛂ⤊஢ᚋ㸪⣧Ỉ࡟ᾐࡋ㸪80Υࡢᜏ ᵴ࡛ 30 min ᣺࡜࠺Ὑίࡋࡓᚋ㸪10 min ㉸㡢ἼὙίࢆ⾜࠸㸪┿✵஝⇱ࡋ㸪poly(SBMA)-g-glass(g-glass-1)ࢆᚓࡓ㸬 ୖグ࡜ྠᵝࡢᡭἲࢆ⏝࠸ BMA ࡜ࡢඹ㔜ྜయ࢞ࣛࢫࡶㄪᩚࡋࡓ㸬ࡇࡇ࡟ SBMA ࡟ᑐࡋ㸪BMA ࢆ SBMA:BMA=99:1
ࡢ ࣔ ࣝ ẚ ࡟ ㄪ ᩚ ࡋ 㸪 poly(SBMA-co-BMA)-g-glass(g-glass-2)ࢆᚓࡓ㸬 JJODVV ⾲㠃ࡢ࢟ࣕࣛࢡࢱࣜࢮ࣮ࢩࣙࣥ
Ỉ࡟ᑐࡍࡿ᥋ゐゅࡣ ᗘㄪ⠇ࢫࢸ࣮ࢪ௜ࡁࡢ Erma ගᏛ♫〇ࢦࢽ࣓࣮࢜ࢱ࣮ᘧ᥋ゐゅ ᐃᶵ G-1 ᆺࢆ⏝࠸࡚ᐃࡋࡓ(23)㸬ࡇࡢ࡜ࡁ㸪⇕㟁ᑐࢆࢫࢸ࣮ࢪୖ࡟᥋ゐࡉࡏ⾲㠃 ᗘࢆィ ࡋ࡞ࡀࡽ±0.1Υ࡛ ᗘㄪ⠇ࡋࡓ㸬ࢫࢸ࣮ࢪᗘࢆ 26Υ࡜ࡋࡓᚋ㸪ᇶᯈୖ࡟ 1 μL ࡢỈ⁲ࢆ⨨࠸ࡓ㸬1min ௨ෆ࡟Ỉ⁲ࡢᕥྑࡢ᥋ゐゅࢆ ᐃࡋࡓ㸬 ᐃࡣィ 5ᅇࡢ࠺ࡕ㸪᭱኱್࠾ࡼࡧ᭱ᑠ್ࢆ㝖࠸ࡓᖹᆒࢆ᥋ゐゅ࡜ࡋࡓ㸬ࡲࡓ ᐃ ᗘࢆ40Υ࡟ୖ᪼ࡉࡏྠᵝࡢ᧯సࢆ⾜࠸㸪－ 50 －40Υ࡛ࡢ᥋ゐゅࡢ ᐃࢆ⾜ࡗࡓ㸬ࡲࡓ㸪㉮ᰝᆺࣉ࣮ࣟࣈ㢧ᚤ㙾(SPM)ࡢ ᐃࡣ Shimadzu 〇 SPM-9500J3 ᆺ㸦࢝ࣥࢳࣞࣂ࣮࡟ Innovation Solutions Bulgaria Ltd㸬〇
Budget sensors Tap300AI-G㸧ࢆ⏝࠸㸪኱Ẽ㞺ᅖẼ୰࡛㸪㉮ᰝ⠊ᅖ㸸30 μm×30 μm㸪㉮ᰝ㏿ᗘ㸸0.5 Hz ࡜ࡋ㸪఩┦᳨ฟࢩࢫࢸ࣒࡟ࡼࡿ⾲㠃ࡢ㧗ప ᐃ࠿ࡽホ౯ࡋࡓ㸬 +H/D ⣽⬊ࡢ᥋╔࡜ቑṪㄪ〇ࡋࡓ g-glass ࡣගᏛࣇ࢕࣒ࣝᅛᐃ⏝㏱᫂୧㠃ࢸ࣮ࣉ HJ-3160W㸦᪥ᮾ㟁ᕤ ᰴ 㸧ࢆࢢࣛࣇࢺ㠃ࡢ⿬㠃࡟㈞ࡾ௜ࡅ㸪g-glass ࢆ 4 ࢙࢘ࣝࡢ࣐ࣝࢳࢹ࢕ࢩࣗ㸦࢙࢘ࣝࢧ࢖ࢬ φ15 mm㸸PS㸧(Nunc)࡟ᅛᐃ໬ࡋࡓ㸬ࢡ࣮ࣜࣥ࣋ࣥ
ࢳ୰࡛ᅛᐃࡋࡓ g-glass ⾲㠃ࢆ࣮࢜ࢺࢡ࣮ࣞࣈ⁛⳦῭ࡢ⁛⳦Ỉ࡟࡚ 800 μL ࡛ 3 ᅇὙίࡋ㸪70%࢚ࢱࣀ࣮ࣝ
(w/v)800 μL ࡛ 1 ᅇὙίࡋࡓ㸬ࡑࡢᚋ㸪ࢡ࣮ࣜࣥ࣋ࣥࢳ୰
ࡢ UV ⁛⳦ⅉୗ⣙ 30 cm ࡢ㊥㞳࡟࣐ࣝࢳࢹ࢕ࢩࣗࡢ⵹ࢆ㛤ࡅ࡚㟼⨨㸪g-glass ⾲㠃ࢆ 1 h UV ↷ᑕࡋ࡚⁛⳦ࡋࡓ㸬⁛⳦ᚋ㸪PBS 800 μL ࡛ 2 ᅇὙίࢆ⾜ࡗࡓ㸬HeLa ⣽⬊ࢆ
10%FCS㸪1%NEAA ྵ᭷ MEM ᇵᆅ࡛ᇵ㣴ࡋࡓ㸬ࡑࡢᚋ㸪HeLa ⣽⬊ࡣ 1 mL ࡢ Trypsin ⁐ᾮ(2.5 mg mL-1)ࢆ
ῧຍࡋ࡚ࡣࡃ㞳ࡋࡓ㸬HeLa ⣽⬊ࡣᾋ㐟⣽⬊⃰ᗘࢆ(3.0×104 cells mL-1)࡟ㄪ〇ᚋ㸪800 μL ࢆୖグ g-glass ୖࢆᅛᐃ໬ࡋࡓ࢙࢘ࣝୖ࡟ຍ࠼㸪࢖࣮ࣥ࢟ࣗ࣋ࢱ࣮
㸦APC-50D㸸࢔ࢫࢸࢵࢡ㸧5%஧㓟໬Ⅳ⣲㞺ᅖẼୗ 30Υ㸪
24 h ᇵ㣴ࡋࡓ㸬+H/D ⣽⬊ࡢ ᗘ่⃭࡟ࡼࡿࡣࡃ㞳࡜⣽⬊ィ 2.4 ⠇࡟ᚑࡗ࡚ HeLa ⣽⬊ࢆᇵ㣴ᚋ㸪g-glass ୖ࡟᥋╔ࢆࡋ࡚࠸࡞࠸ HeLa ⣽⬊ࢆ㝖ཤࡍࡿࡓࡵ㸪PBS ࢆ⏝࠸࡚㠀᥋╔⣽⬊ࢆ 1 ᅇὙίࡋࡓᚋ㸪ಽ❧ᆺ࣮ࣝࢳࣥ㢧ᚤ㙾(CKX31㸸OLYMPUS)࡛ほᐹࡋ㸪㢧ᚤ㙾ࢹࢪࢱ࣓ࣝ࢝ࣛ
(DP72-BSW)ࢆ⏝࠸࡚᧜ᙳࡋࡓ㸬ࡑࡢᚋ㸪 ᗘ่⃭࡟ࡼࡿࡣࡃ㞳ࢆ⾜࠺ࡓࡵ㸪ணࡵ㸪 ᗘࢆ 37Υ࡟タᐃࡋ࡚࠾࠸
ࡓࢧ࣮ࣔࣉ࣮ࣞࢺ(FTP-28190㸸AZ ONE)࡟ g-glass ࢆᅛᐃࡋ࡚࠸ࡿ࣐ࣝࢳࢹ࢕ࢩࣗࢆ 2 h 㟼⨨ࡋࡓ㸬㟼⨨ᚋ㸪800
μL ࡢ PBS ࢆ⏝࠸㸪3 ᅇὙίࡋ࡚ࡣࡃ㞳⣽⬊ࢆ㝖ཤࡋ㸪࢞ࣛࢫୖ࡟ṧᏑࡍࡿ᥋╔⣽⬊ᩘࢆಽ❧ᆺ࣮ࣝࢳࣥ㢧ᚤ㙾(CKX31㸸OLYMPUS)࡛ほᐹࡋ㸪㢧ᚤ㙾ࢹࢪࢱ࣓ࣝ࢝ࣛ
(DP72-BSW)ࢆ⏝࠸࡚᧜ᙳࡋࡓ㸬⣽⬊ᩘࡢィ ࡣ㸪ಽ❧ᆺ࣮ࣝࢳࣥ㢧ᚤ㙾(CKX31㸸OLYMPUS)ࡢほᐹ࠿ࡽぢ✚ࡶࡗࡓ㸬⣽⬊ࡣ␗࡞ࡿ 5 ⟠ᡤࡢ㢧ᚤ㙾ど㔝ࢆࢹࢪࢱࣝ⏬
ീ࡜ࡋ࡚ಖᏑᚋ㸪⣽⬊ᩘࢆィ ࡋࡓ㸬ࡑࡢᖹᆒ್ࢆ᥋╔⣽⬊ᩘ࡜ࡋࡓ㸬୍᪉㸪ຍ ࡟ࡼࡿࡣࡃ㞳᧯సᚋ㸪ୖ⃈ࡳࢆྲྀࡾ㝖ࡁ㸪ṧᏑࡍࡿ᥋╔⣽⬊ᩘࢆྠᵝ㸪ィ ࡋࡓ㸬ࡣࡃ㞳⋡(%)ࡣ᥋╔⣽⬊ᩘ࡜ࡢᕪ࠿ࡽ⟬ฟࡋࡓ㸬 ⤖ᯝ࡜⪃ᐹ 3RO\ 6%0$ ࡜ SRO\ 6%0$FR%0$ ࢢࣛࣇࢺ໬࢞ࣛࢫJJODVV ࡢㄪ〇࡜⾲㠃࢟ࣕࣛࢡࢱࣜࢮ࣮ࣥࣙࣥ⣽⬊ᇵ㣴࠾ࡼࡧࡣࡃ㞳ᇶᮦ࡜ࡋ࡚ࡢᛂ⏝ࡉࡽ࡟ࡣ CW࣮ࣞࢨ࣮↷ᑕᶵࢆ⏝࠸ࡓᒁᡤⓗ࡞ຍ ࡟ࡼࡿ⣽⬊ࡢ㑅ᢥⓗ࡞ࡣࡃ㞳࣭ᅇ཰ࢆ┠ⓗ࡜ࡋ㸪UCST ࢆ♧ࡍ poly(SBMA)࠾ࡼࡧ poly(SBMA-co-BMA)-g-glass ࡢㄪ〇ࢆ⾲㠃㛤ጞࣛࢪ࢝ࣝ㔜ྜἲ࡟ࡼࡾヨࡳࡓ㸬⾲㠃㛤ጞ㔜ྜᚋࡢ࢞ࣛࢫ⾲㠃ࡢ poly(SBMA)࡜ poly(SBMA-co-BMA)ࡢಟ㣭ࢆ☜ㄆࡍࡿࡓࡵ㸪኱Ẽ㞺ᅖẼୗ࡛ࡢ⾲㠃ࡢ SPM
ᐃ(30μm×30 μm)ࡢ⤖ᯝࢆ Fig.1 ࡟♧ࡍ㸬ᮍฎ⌮࢞ࣛࢫ࡛ࡣพฝࡀ↓ࡃࣇࣛࢵࢺ࡛࠶ࡿࡇ࡜࡟ᑐࡋ㸪poly(SBMA)࡜
poly(SBMA-co-BMA)ࢆࢢࣛࣇࢺ㔜ྜࡋࡓ g-glass ࡛ࡣࡑࢀࡒࢀ㸪ࡇࢀࡽ࡟࣏࣐࣮ࣜ⏤᮶࡜⪃࠼ࡽࢀࡿࢼࣀࢫࢣ࣮࡛ࣝࡢࣈࣛࢩ≧ࡢพฝࡀ⾲㠃ୖ࡟ほᐹࡉࢀࡓ㸬ḟ࡟㸪

Fig.1 SPM image of glasses;(b)g-glass-1, and (c) g-glass-2.

A-1(a)Untreated,B-1

Untreated

A-2
g-glass-1

A-3
g-glass-2

Fig.2 Phase-contrast microscope observation of HeLa
cells attached glasses; (A)30Υ and (B) 37Υ.
g-glass ࡢỈ࡟ᑐࡍࡿ᥋ゐゅ(θ)ࢆ ᐃࡋࡓ⤖ᯝ㸪ࢫࢸ࣮ࢪᗘ 26 Υ ࡛ ࡣ ᮍ ฎ ⌮ ࢞ ࣛ ࢫ ࡢ θ=21 r ࡟ ᑐ ࡋ ࡚poly(SBMA)ࢆಟ㣭ࡋࡓ g-glass-1 ࡛ࡣ θ=54r࡜࡞ࡾ
poly(SBMA)ᅛᐃ໬࡟ࡼࡾ θ ࡀቑ኱ࡋ㸪࢞ࣛࢫ⾲㠃ࡀ␯Ỉᛶ࡬࡜ኚ໬ࡋࡓࡇ࡜ࢆㄆࡵࡓ㸬ࡲࡓ㸪BMA ࢆඹ㔜ྜࡋࡓg-glass-2 ࡣྠᵝ࡟ θ=58r࡜࡞ࡾ␯Ỉᛶࡢቑຍࢆㄆࡵࡓ.
θ ࡣ g-glass-1 ࡼࡾ g-glass-2 ࡀቑຍࡋ㸪BMA ࢭࢢ࣓ࣥࢺ－ 51 －࠶ࡿ࡜⪃࠼ࡽࢀࡿ㸬100% of exfoliationࡢᑟධ࡟ࡼࡗ࡚㸪⾲㠃ࡢ␯Ỉᛶࡀቑ኱ࡋࡓ㸬୍᪉㸪ࢫࢸ࣮ࢪ ᗘࢆ 40Υ࡟ୖࡆ ᐃࡋࡓ ǉ ࡣᮍฎ⌮࢞ࣛࢫࡣኚ໬ࡋ࡞࠸ࡢ࡟ᑐࡋ㸪g-glass-1 ࡛ࡣ ǉ=45r㸪
g-glass-2 ࡛ࡣ ǉ=49r࡜ ᗘୖ᪼࡟ࡼࡗ࡚㸪⾲㠃ࡢ⃿ࢀᛶࡀቑ኱ࡋ࡚ぶỈᛶ࡬࡜ኚ໬ࡋࡓ㸬ࡇࢀࡣ㸪࢞ࣛࢫ⾲㠃࡟ࢢࣛࣇࢺࡋࡓ poly(SBMA)࠾ࡼࡧ poly(SBMA-co-BMA)ࡀ
UCST ௨ୖ࡜࡞ࡾ㸪ࡑࢀࡒࢀࡢ࣏࣐࣮ࣜ㙐ࡀࢢࣟࣅ࣮ࣗࣝ≧࠿ࡽࣛࣥࢲ࣒ࢥ࢖ࣝ≧࡬࡜ኚ໬ࡋ㸪⾲㠃ࡢ⃿ࢀᛶࡀ␯Ỉᛶ࠿ࡽぶỈᛶ࡬࡜ኚ໬ࡋࡓࡓࡵ࡜⪃࠼ࡽࢀࡿ㸬 JJODVV ⾲㠃࡛ࡢ⣽⬊ᇵ㣴࡜ ᗘ่⃭࡟ࡼࡿࡣࡃ㞳
3.1 ⠇࡛ ᗘ่⃭࡟ࡼࡾぶ࣭␯Ỉᛶࡀኚ໬ࡋ㸪SPMᐃ࡟ࡼࡾ⾲㠃ᛶ≧ࡢኚ໬ࢆㄆࡵࡓ g-glass ୖࡢ⣽⬊ᇵ㣴࡞ࡽࡧ࡟ຍ ࡟ࡼࡿࡣࡃ㞳ᛶ⬟ࢆ▱ࡿࡓࡵ㸪ᮍฎ⌮࢞ࣛࢫ࡜ g-glass ⾲㠃࡛ࡢ HeLa ⣽⬊ࢆ⏝࠸ࡓ⣽⬊ᇵ㣴࡜ຍ࡟ࡼࡿ ᗘ่⃭ࡣࡃ㞳ࢆ᳨ウࡋࡓ㸬4 well ࡢᇵ㣴ࢩ࣮ࣕࣞ࡟┤ᚄ 12 mm ࡢ g-glass ࠾ࡼࡧᮍฎ⌮࢞ࣛࢫࢆ wellୖ࡟㏱᫂୧㠃ࢸ࣮ࣉ࡛㈞ࡾ௜ࡅࡓ㸬UV ⁛⳦ᚋ㸪3.0×104
cells mL-1 ࡢ HeLa ⣽⬊ࢆ᧛✀ᚋ㸪24 h ⤒㐣ᚋࡢ఩┦ᕪ㢧ᚤ㙾෗┿ࢆ Fig.2 ࡟♧ࡍ㸬ᅗ࠿ࡽ 30Υ࡛ HeLa ⣽⬊g-glass-1,2 ࠸ࡎࢀࡶ࢞ࣛࢫᇶᯈୖ࡜ྠᵝ࡟᥋╔࣭ఙᒎࡋቑṪྍ⬟࡛࠶ࡗࡓ㸬ḟ࡟㸪ຍ ࡟ࡼࡾ⾲㠃ࡀ␯Ỉᛶ࠿ࡽぶỈᛶ࡬ࡢᛂ⟅ࢆㄆࡵࡓ g-glass ࢆ⏝࠸࡚㸪᥋╔⣽⬊ࡢࡣࡃ㞳ᐇ㦂ࢆ⾜ࡗࡓ㸬ᇵ㣴ᚋ㸪PBS(25Υ)Ὑίࢆ 1 ᅇ⾜࠸㸪ᇶᯈ࡬ࡢᮍ᥋╔ࡢ⣽⬊ࢆྲྀࡾ㝖ࡁ㸪37Υࡢ࣍ࢵࢺࣉ࣮ࣞ
ࢺୖ࡟ g-glass ࢆ᥋╔ᅛᐃࡋ࡚࠸ࡿ 4 well ࣐ࣝࢳࢹ࢕ࢵࢩࣗࢆ⛣ࡋ㸪2 h 㟼⨨ࡋࡓ㸬ḟ࡟㸪PBS(25Υ)࡛ 3 ᅇὙίࡋ㸪ࡣࡃ㞳ࢆ⾜ࡗࡓ㸬ຍ ࡟ࡼࡿ HeLa ⣽⬊ࡢࡣࡃ㞳⋡ࢆᮍฎ⌮࢞ࣛࢫ࡜ేࡏ࡚㸪᥋╔ṧᏑࡍࡿ⣽⬊ᩘ࠿ࡽぢ✚ࡶࡗࡓࡣࡃ㞳⣽⬊ᩘࡢ๭ྜࢆ Fig.3 ࡟♧ࡍ㸬g-glass-1 ࡣࡣࡃ㞳⋡⣙ 40㸣࡜ᮍฎ⌮࢞ࣛࢫ(ࡣࡃ㞳⋡ 24%)ࡼࡾࡶ
15%⛬ᗘୖ᪼ࡋࡓ㸬୍᪉㸪g-glass-2 ࡢࡣࡃ㞳⋡(14%)ࡣ
g-glass-1 ࠾ࡼࡧᮍฎ⌮࢞ࣛࢫࡼࡾࡶࡣࡃ㞳⋡ࡣపୗࡋࡓ㸬୍᪉㸪UCST ࢆ♧ࡍ poly(SBMA)ࢢࣛࣇࢺ㙐ࡢᅛᐃ໬࡟
ࡼࡾ㸪UCST ௨ୗࡢ ᗘ࡛᥋╔ࡋࡓ HeLa ⣽⬊ࡣ UCST௨ୖ࡟ຍ ࡍࡿࡇ࡜࡟ࡼࡾࡣࡃ㞳ࢆಁ㐍ࡍࡿຠᯝࢆㄆࡵࡓ㸬୍᪉㸪poly(SBMA-co-BMA)ᅛᐃ໬࢞ࣛࢫ࡛ࡣᮍฎ⌮࢞ࣛࢫࡼࡾࡣࡃ㞳⋡ࡣపୗࡋ㸪ᮏᛶ⬟ࡀప࠸㸬୧ᛶ࢖࢜ࣥᆺ SBMA 㙐ࡢ♧ࡍ UCST ࡣ࣏࣐࣮ࣜ㙐㛗ཬࡧ፹యࡢ
ሷ➼࡟኱ࡁࡃᙳ㡪ࢆཷࡅࡿ࡜⪃࠼ࡽࢀࡿ㸬ࡲࡓ ᗘᛂ⟅ᛶ࣏࣐࣮ࣜ㙐ᅛᐃ໬⾲㠃ࡣ࣏࣐࣮ࣜ㙐ࡢᐦᗘࡸศᏊ㔞࡟኱ࡁࡃᙳ㡪ࢆཷࡅࡿࡇ࡜࠿ࡽ(24) 㸪⣽⬊᥋╔ཬࡧࡣࡃ㞳࡟᭱㐺࡞ᛶ≧࡟ᅛᐃ໬࣏࣐࣮ࣜ㙐ࢆㄪᩚࡍࡿᚲせࡀ࠶ࡿ࡜⪃࠼ࡽࢀࡿ㸬ࡲࡓ㸪g-glass-2 ࡛ࡣ㸪ࡣࡃ㞳⋡ࡀᮍฎ⌮࢞ࣛࢫࡼࡾࡶపࡃ㸪ඹ㔜ྜࡋࡓ BMA ࢭࢢ࣓ࣥࢺ࡟ࡼࡿ␯Ỉᛶࡢᙳ㡪➼ࡀ⪃࠼ࡽࢀࡿࡓࡵ㸪UCST ᗘࡢࡳ࡞ࡽࡎᅛᐃ໬᫬ࡢ⾲㠃ࡢぶ࣭␯Ỉᛶ࡟ࡘ࠸࡚ࡶ᳨ウࡍࡿᚲせࡀ

9080
706050

He L a cells

403020100
Untreatedg-glass-1g-glass-2

Fig.3 Number of exfoliated HeLa cells at 37Υ.
 ࡲ࡜ࡵ
ձSPM ᐃ࠾ࡼࡧ㸪Ỉࡢ᥋ゐゅࡢ ᐃ⤖ᯝ࡟ࡼࡾ㸪⾲㠃㛤ጞࣛࢪ࢝ࣝ㔜ྜἲ࡟ࡼࡗ࡚࢞ࣛࢫᇶᯈ⾲㠃࡟
poly(SBMA)࠶ࡿ࠸ࡣ poly(SBMA-co-BMA)ࡢᅛᐃ໬ࢆ
ㄆࡵࡓ㸬
ղpoly(SBMA)࠶ࡿ࠸ࡣ poly(SBMA-co-BMA)ࢆᅛᐃ໬
ࡋࡓ࢞ࣛࢫࡣ 30Υ࠿ࡽ 40Υࡢຍ ࡟ࡼࡾ᥋ゐゅࡀపୗࡋ⾲㠃ࡀ␯Ỉᛶ࠿ࡽぶỈᛶ࡟ኚ໬ࡍࡿ UCST ᛶࢆ♧ࡋࡓ㸬ճ HeLa ⣽ ⬊ ࢆ ⏝ ࠸ ࡓ ᥋ ╔ ࣭ ࡣ ࡃ 㞳 ヨ 㦂 ࡛ ࡣ 㸪poly(SBMA)ࡲࡓࡣ poly(SBMA-co-BMA)ಟ㣭࢞ࣛࢫᇶᯈࡣ㸪30Υ࡛ࡣ⣽⬊ࡀ࢞ࣛࢫᇶᯈ⾲㠃࡟᥋╔࣭ቑṪࡋࡓ㸬
poly(SBMA)ᅛᐃ໬࢞ࣛࢫࡣ 37Υ࡬ ᗘࢆୖ᪼ࡍࡿࡇ࡜࡟ࡼࡿ⣽⬊ࡢࡣࡃ㞳⋡ࡢྥୖࢆㄆࡵࡓ㸬௨ୖࡢ⤖ᯝ࠿ࡽ㸪UCST ࢆ♧ࡍ࣏࣐࣮ࣜࡢᅛᐃ໬࡜㸪ຍ࡟ࡼࡿ ᗘ่⃭࡟ࡼࡾ⣽⬊ࡣࡃ㞳ࡀྍ⬟࡛࠶ࡿ࡜⪃࠼ࡽࢀࡿ㸬᥋╔⣽⬊ࡢࡣࡃ㞳⋡ࢆྥୖࡉࡏࡿࡓࡵ࡟ࡣ㸪ᅛᐃ໬࣏࣐࣮ࣜࡢ㙐㛗㸪ᐦᗘ㸪⤌ᡂࡉࡽ࡟ࡣ㸪ࡣࡃ㞳᫬ࡢᗘ➼ࡢࡉࡽ࡞ࡿ᭱㐺໬ࡀᚲせ࡛࠶ࡿ㸬ຍ ࡟ࡼࡿࡣࡃ㞳ᛶ⬟ࡢ☜❧࡟ࡼࡗ࡚㸪UCST ᆺ࣏࣐࣮ࣜᅛᐃ໬ ǍAy ࡜ CW ࣮ࣞࢨ࣮↷ᑕᶵࢆ⏝࠸ࡓࢩࢫࢸ࣒࡟ࡼࡗ࡚┠ⓗ⣽⬊ࡢ㑅ᢥⓗ࡞ࡣࡃ㞳࣭ᅇ཰ࢶ࣮ࣝࡢస〇࡟ᛂ⏝ࡀ࡛ࡁࡿ࡜⪃࠼ࡽࢀࡿ㸬ㅰ㎡ᮏ◊✲ࡢ୍㒊ࡣ⤒῭⏘ᴗ┬ᆅᇦ࢖ࣀ࣮࣋ࢩࣙࣥ๰ฟ㛤Ⓨ஦ᴗཬࡧᡓ␎ⓗᇶ┙ᢏ⾡㧗ᗘ໬᥎㐍஦ᴗࡢᨭ᥼࡟ࡼࡿࡶࡢ࡛࠶ࡾ㸪ࡇࡇ࡟ㅰពࢆ⾲ࡋࡲࡍ㸬ཧ⪃ᩥ⊩(1) S. Yamanaka et al., Cell, 126 (4),(2006), 663－ 52 －(2) M. Oshimura et al., PLoS ONE, 10(6), (2011),e25961(3) M. Katoh et al., BMC Biotechnology , 10, (2010),
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