近畿大学次世代基盤技術研究所報告 Vol. 6（2015）55－59 UCSTᆺࣀࢽ࢜ࣥ⣔࣏࣐࣮ࣜࢆ⾲㠃ಟ㣭ࡋࡓ࢞ࣛࢫᇶᯈࡢ ㄪ〇HeLa⣽⬊ࡢ ᗘ่⃭ࡣࡃ㞳 ᇛ ᫂*1㸪ఀ⸨ *1㸪ᒣ⏣ ᗣᯞ*2㸪ⓑ▼ ᾈᖹ*2 Preparation of UCST types of nonion polymer modified glass and thermal stimuli-exfoliation of HeLa cells on the substrate Akinori IMAJO*1㸪Taiji ITO*1㸪Yasue YAMADA*2 㸪and Kohei SHIRAISHI*2 Poly(acrylamide-co-acrylonitrile)[poly(AAm-co-AN)]with upper critical solution temperature (UCST) was grafted onto a glass substrate by surface initiated atom transfer radical polymerization(SI-ATRP). From the scanning probe microscope(SPM) measurements , poly(AAm-co-AN) brushes were observed on the glass substrate(g-glass). The contact angle(θ) value of g-glass to water at 26°C increased from θ=21°(the untreated glass) to θ=69°. On the other hand, that of g-glass at 40°C decreased from θ=69° to θ=65°. After being HeLa cells attached and proliferated on the g-glass at 30°C under UCST of poly(AAm-co-AN) burshes, the HeLa cells attached were thermally exfoliated by increasing culture temperature over 37°C for 30 min in phosphate buffered saline(pH7.4). The % of mechanical and thermal stimuliexfoliation of attached HeLa cells on the g-glass was about 90% despite of about 10% on untreated glass. Keywords: Poly(acrylamide-co-acrylonitrile), Upper critical solution temperature (UCST), Surface initiated atom transfer radical polymerization(SI-ATRP), Thermal exfoliation, HeLa cells ࡣࡌࡵ iPS ⣽⬊(induced pluripotent stem cells)ࡸ ES ⣽⬊ (embryonic stem cells)࠸ࡗࡓከ⬟ᛶᖿ⣽⬊ࡢᐇ⏝ ࡼࡾ㞴ᝈ➼ᑐࡍࡿ᪂ࡓ࡞⒪ἲࡢ㛤Ⓨࡸ⏕་⒪ ࡢᛂ⏝ࡲࡓ⸆ࢫࢡ࣮ࣜࢽࣥࢢࡀᮇᚅࡉࢀ࡚࠸ࡿ㸬ࡋ ࡋ㸪iPS ⣽⬊➼ࡢከ⬟ᛶᖿ⣽⬊ࡢᐇ⏝ࡣ㸪స〇ຠ ⋡ࡢྥୖࡸ⣽⬊ᶵ⬟ࡢ⢭ᐦデ᩿㸪⏝⣽⬊ࡢ㝖ཤ࠾ࡼࡧ ┠ⓗ⣽⬊ࡢࡳࡢ㠀くⓗ࡞ᅇᢏ⾡ࡢ㛤Ⓨ➼ࡢᵝࠎ࡞ㄢ 㢟ࡀ࠶ࡿࡉࢀࡿ㸬≉㸪⣽⬊ࡢᇵ㣴ཬࡧᅇᢏ⾡ࡢ㛤 Ⓨࡣ㸪ከ⬟ᛶᖿ⣽⬊ࡢࡳ࡞ࡽࡎ╔ᛶ⣽⬊ࡢ◊✲࠾࠸ ࡚ࡶ㔜せ࡞ㄢ㢟࡛࠶ࡿ㸬ࡇࡢㄢ㢟ゎỴࡣ㸪⣽⬊᧯స ಀࡿ⣲ᮦࡸ࿘㎶ᶵჾ࠶ࡿ࠸ࡣჾᲔࡶᚲせ࡛࠶ࡾ㸪ࣟ࣎ࢸ ࢡࢫᢏ⾡ࢆే⏝ࡋࡓ⮬ືࢩࢫࢸ࣒ࢆ⏝࠸ࡓデ᩿ཬࡧ ᅇࡢࣁࢫ࣮ࣝࣉࢵࢺ࣭పࢥࢫࢺ࣭⨨ᑠᆺ➼ ࡢㄢ㢟ࡶ࠶ࡿ㸬ࡲࡓ㸪⌧ᅾ⏝ࡉࢀ࡚࠸ࡿࣇ࣮ࣟࢭࣝࢯ ࣮ࢱ➼ࡢ⣽⬊ࡢデ᩿ཬࡧ㑅ᢥⓗ࡞ᅇ⨨ࡣ๓ฎ⌮᧯స ➼ࢆྵࡵ࡚↹㞧࡛࠶ࡿࡉࢀ㸪㧗⢭ᗘ࡛⣽⬊ࢆᅇࡍࡿ ࡓࡵࡣ⇍⦎ࡋࡓᢏ⾡⪅ࡀᚲせࡉࢀࡿ➼㸪ࣂ࢜་⒪ ᨭᶵჾࡋ࡚ᬑཬࡍࡿࡣከࡃࡢㄢ㢟ࡀṧࡉࢀ࡚࠸ࡿ㸬 ᚑࡗ࡚㸪Ᏻ࡛࠶ࡾ㸪Ᏻ౯࡛ࣂ࢜⣔ᢏ⾡⪅ࡢ⇍⦎ࡋࡓ ᢏ⾡ࢆᚲせࡋ࡞࠸⡆౽࡞᧯స࡛⾜࠼ࡿࢶ࣮ࣝࡢ㛤Ⓨࡀ ồࡵࡽࢀ࡚࠸ࡿ㸬 ⌧ᅾ㸪⣽⬊デ᩿ࢆከ᳨యྠ㸪ࡘࣁࢫ࣮ࣝࣉࢵ ࢺ⾜࡞࠼ࡿࢶ࣮ࣝࡋ࡚⣽⬊࣐ࢡࣟࣞ(μAy)ࡀ ὀ┠ࡉࢀ࡚࠸ࡿ(1),(2)㸬≉㸪μAy ୖ⣽⬊ࢆ➼㛫㝸㓄 ⨨ࡍࡿᡭἲࡣ㸪ᶵ⬟ゎᯒࡢࡓࡵࡢࢶ࣮ࣝࡋ࡚ὀ┠ࡉࢀ ࡚࠾ࡾ㸪⸆ࡢࡓࡵࡢ⸆ࢫࢡ࣮ࣜࢽࣥࢢ(3)~(5)㸪་⸆ရ ࡸᏛ≀㉁࡞ࡢ᭷⏝ᛶࡸᏳᛶࡢホ౯(6)~(8)㸪⣽⬊ࡢศ ཎ✏ཷ 2015 ᖺ 5 ᭶ 8 ᪥ *1 ㏆␥ᏛᏛ㝔 ࢩࢫࢸ࣒ᕤᏛ◊✲⛉ ࢩࢫࢸ࣒ᕤᏛᑓᨷ 㸦ࠛ739-2116 ᮾᗈᓥᕷ㧗ᒇ࠺ࡵࡢ㎶ 1 ␒㸧 E-mail [email protected] *2 ㏆␥ᏛᏛ㝔 ࢩࢫࢸ࣒ᕤᏛ◊✲⛉ ࢩࢫࢸ࣒ᕤᏛᑓᨷ㸪ᕤᏛ㒊 Ꮫ⏕ᕤᏛ⛉ ᩍᤵ㸪ḟୡ௦ᇶ┙ᢏ⾡◊✲ᡤ ᩍᤵ 㸦ࠛ739-2116 ᮾᗈᓥᕷ㧗ᒇ࠺ࡵࡢ㎶ 1 ␒㸧 E-mail [email protected] 㐃⤡ඛ㸸ⓑ▼ᾈᖹ㸦◊✲௦⾲⪅㸧 － 55 － ㄏᑟᅉᏊࡸ㑇ఏᏊ⩌ࡢࢫࢡ࣮ࣜࢽࣥࢢ(9)~(11)࡞ ǍAy ࡢྍ⬟ᛶࡣࡁ࠸㸬ࡲࡓ㸪⣽⬊ᶵ⬟ࡢデ᩿࡛ࡣ㸪ᚑ᮶ࡲ ࡛ࡣከࡃࡢ⣽⬊ࡽ୍ᣓ࡛ᚓ࡚࠸ࡓሗࢆ㸪୍ಶ࠶ࡿ࠸ ࡣᩘಶࡢ㝈ࡽࢀࡓ⣽⬊ẖᚓࡽࢀࡿ㸬⣽⬊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 ǍAy ࢆ⏝࠸࡚㸪ಶ⣽⬊ࡢデ᩿ཬࡧ㠀く ⓗࡘ㑅ᢥⓗᅇࡋᚓࡿᢏ⾡ࡢ㛤Ⓨࢆ⾜࡞ࡗ࡚࠸ࡿ (12),(13)㸬ࡇࡢࡁ㠀くⓗ࡞⣽⬊ᅇᶵ⬟ࢆ࠼ࡿࡓࡵ㸪 ᗘᛂ⟅ᛶ࣏࣐࣮ࣜࡢᅛᐃࢆ᳨ウࡋ࡚࠸ࡿ㸬ୗ㝈⮫⏺ ඹ⁐ ᗘ(LCST)ᆺ ᗘᛂ⟅ᛶ࣏࣐࣮ࣜࡣ㸪⁐ᾮ୰ࡢ ᗘ ኚ࡛ぶ࣭Ỉᛶࡀ㸪┦㌿⛣ ᗘࢆቃప 㸦ぶỈᛶ㸧 я㧗 㸦Ỉᛶ㸧ྍ㏫ⓗኚࡍࡿ㸬LCST ᆺ࣏࣐ࣜ ࣮ࢆ⾲㠃ᅛᐃࡋࡓ⣲ᮦࡣ㸪᥋╔⣽⬊ࡢᇵ㣴 ᗘࢆప ୗࡉࡏ㠀くⓗ⣽⬊ᅇࡍࡿ᪉ἲࡋ࡚ᐇ⏝ࡉࢀ࡚ ࠾ࡾ(14),(15)㸪ǍAy ࢆ⏝࠸ࡓ⣽⬊ࡢ㑅ᢥⓗᅇࡢᛂ⏝ࢆ㐍 ࡵ࡚࠸ࡿ㸬୍᪉㸪LCST ᆺ࣏࣐࣮ࣜᅛᐃ ǍAy ୖ࡛ࡢ㑅 ᢥⓗ࡞ᅇ࡛ࡣ㸪⨨ࡀ」㞧࡞ࡾ ǍAy ࢫ࣏ࢵࢺࡢᒁᡤ ࢆ෭༷ࡍࡿ㸪࣮ࣞࢨ࣮ග࡛⏝⣽⬊ࢆணࡵ㝖ཤࡋࡓࡢ ࡕ㸪ǍAy యࢆ෭༷ࡋ࡚┠ⓗ⣽⬊ࢆᅇࡍࡿ᪉ἲࢆ㛤Ⓨ ࡋ࡚࠸ࡿ(14)㸬ᮏ ǍAy ࡛ࡣ┠ⓗ⣽⬊ࡀᴟᑡᩘࡢሙྜᛂ⏝ ࡀࡋࡃ࠸ࡇࡽ㸪LCST ᆺ࣏࣐࣮ࣜ㏫ࡢ㧗 ࡛ぶ Ỉᛶࢆ♧ࡋప ࡛Ỉᛶࢆ♧ࡍୖ㝈⮫⏺ඹ⁐ ᗘ (UCST)ᆺ ᗘᛂ⟅ᛶ࣏࣐࣮ࣜ(16),(17)ࢆ⏝࠸㸪ǍAy ࢫ࣏ࢵ ࢺᅛᐃࡋ࡚㸪㧗⢭ᗘ࡞⨨Ỵࡵ CW ࣮ࣞࢨ࣮ග↷ᑕ ࡼࡿᒁᡤຍ ࡼࡿ㑅ᢥⓗ࡞ᅇࡢᇶ♏ⓗ࡞▱ぢࢆᚓࡿ ࡓࡵ㸪ǍAy ࢫ࣏ࢵࢺྠࡌ࢞ࣛࢫ㠃 UCST ᆺ࣏࣐࣮ࣜ ࢆ⾲㠃㛤ጞཎᏊ⛣ືࣛࢪ࢝ࣝ㔜ྜἲ(18)ࡼࡿᅛᐃࢆヨ ࡳࡓ㸬ࡇࡇ࡛ࡣ㸪⣽⬊ᇵ㣴ᇵᆅ⤌ᡂࡼࡿ UCST ࡢ ᙳ㡪ࡀᑡ࡞࠸⪃࠼ࡿࣀࢽ࢜ࣥᵓ㐀ࡢࢡ࣑ࣜࣝࢻ ࢡࣜࣟࢽࢺࣜࣝࡢඹ㔜ྜయ࡛࠶ࡿ Poly(acrylamide-co- acrylonitrile) [poly(AAm-co-AN)](19) ࢆ 㑅 ᢥ ࡋ 㸪 poly(AAm-co-AN)ᅛᐃ࢞ࣛࢫ(g-glass)ୖ࡛ HeLa ⣽⬊ࢆᇵ 㣴ᚋ࠾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ᮾிᡂᰴ〇㸧 㸪 N,N-ࢪ࣓ࢳ࣒࣑ࣝ࣍ࣝࢻ㸦⬺Ỉ㸧(DMF)㸦ග⣧⸆ᰴ 〇㸧 㸪ࢺ࢚ࣜࢳ࣑ࣝࣥ㸦ග⣧⸆ᰴ〇㸧 㸪ࢡࣜࣝ ࣑ࢻ (AAm)㸦ග⣧⸆ᰴ〇㸧㸪ࢡࣜࣟࢽࢺࣜࣝ (AN) 㸦ᮾிᡂᰴ〇㸧㸪4-ࢪ࣓ࢳ࣑ࣝࣀࣆࣜࢪࣥ (DMAP) 㸦ග⣧⸆ᰴ㸧 㸪 ⮯㖡(Ϩ) (CuBr(Ϩ))㸦ග⣧⸆ᰴ㸧 㸪 ࢪ࣓ࢳࣝࢫࣝ࣍࢟ࢩࢻ (DMSO)㸦ග⣧⸆ᰴ〇㸧㸪 N,N,N',N",N" -࣌ࣥࢱ࣓ࢳࣝࢪ࢚ࢳࣞࣥࢺ࣑ࣜࣥ (PMDETA)㸦ග⣧⸆ᰴ〇㸧㸪L-ࢫࢥࣝࣅࣥ㓟 㸦 ග⣧⸆ᰴ〇㸧㸪2-ࣈࣟࣔࢯࣈࢳࣜࣝࣈ࣑ࣟࢻ (BiBB) 㸦ᮾிᡂᰴ〇㸧ࡣᕷ㈍ရࢆࡑࡢࡲࡲ⏝࠸ࡓ㸬࣓ࢱࣀ ࣮ࣝ㸪ࢺ࢚ࣝࣥࡢ⁐፹ࡶᕷ㈍ရࢆࡑࡢࡲࡲ⏝࠸ࡓ㸬⣧Ỉ ࡣMILLIPORE 〇MILL-Q Synthesis A10㉸⣧Ỉ〇㐀 ⨨ࢆ⏝࠸࡚⢭〇ࡋ࡚⏝ࡋࡓ㸬࢝ࣂ࣮࢞ࣛࢫࡣ Microscope Cover Glass(Ǘ=12 mm)(Fisher Scientific 〇ࢆࡑࡢࡲࡲ⏝࠸ࡓ㸬Minimum Essential Medium(MEM)㸦GIBCO 〇㸧 㸪∵⫾ඣ⾑Ύ(FCS) (GIBCO 〇ࡣᖖἲᚑࡗ࡚ㄪᩚࡋ࡚⏝࠸ࡓ㸬㠀ᚲ㡲࣑ࣀ㓟 (NEAA)ࡣࡑࡢࡲࡲ⏝࠸ࡓ㸬ࣜࣥ㓟⦆⾪⏕⌮㣗ሷỈ (PBS)(pH7.4)ࡣᖖἲᚑࡗ࡚ㄪᩚࡋ࡚⏝࠸ࡓ㸬0.25㸣 Trypsin ࡣᖖἲᚑࡗ࡚ㄪᩚࡋ࡚⏝࠸ࡓ㸬⁛⳦Ỉࡣ⣧Ỉ ࢆ࣮࢜ࢺࢡ࣮ࣞࣈ⁛⳦ࡋ࡚⏝࠸ࡓ㸬 $$P$1 ඹ 㔜 ྜ య ࢢ ࣛ ࣇ ࢺ ࢞ ࣛ ࢫ >SRO\$$PFR$1JJODVV@ࡢㄪ〇 ࢞ࣛࢫ〇ࢩ࣮ࣕࣞࢆ⏝࠸㸪ࣆࣛࣥࣁ⁐ᾮ[30㸣㐣㓟Ỉ ⣲/18 M ◲㓟=1/3(v/v)]1.6 mL ࢝ࣂ࣮࢞ࣛࢫࢆධࢀ 1 h ᾐₕࡋ㸪Ỉ࡛࢝ࣂ࣮࢞ࣛࢫࢆὙίࡋࡓᚋ㸪࢚࣮ࢥࣥࣉ ࣞࢵࢧ࣮࡛✵Ẽࢆ྿ࡁࡅ࡚⇱ࡋࡓ㸬Ὑίᚋ㸪0.179 g (1 mmol) 3-࣑ࣀࣉࣟࣆࣝࢺ࣓ࣜࢺ࢟ࢩࢩࣛࣥࡢ DMF ⁐ᾮ 2 mL ࡀධࡗࡓ࢞ࣛࢫ〇ࢫࢡ࣮ࣜࣗ⟶࢝ࣂ࣮࢞ࣛ ࢫࢆධࢀ㸪60Υ㸪3 h ᛂࡋࡓ㸬ࡑࡢᚋ㸪࣓ࢱࣀ࣮࡛ࣝ ⾲㠃ࢆὙίࡋ㸪80Υ㸪4 h ⇱ჾ࡛↝ࡁࡅ࣭⇱ࢆ⾜ ࡗࡓ㸬↝ࡁࡅᚋ㸪࣓ࢱࣀ࣮࡛ࣝ㉸㡢ἼὙί 10 min ⾜ࡗ ࡓ㸬࢞ࣛࢫ〇ࢫࢡ࣮ࣜࣗ⟶ࢺ࢚ࣜࢳ࣑ࣝࣥ 0.0174 g (0.146 mmol) DMAP 0.00176 g (0.0144 mmol)ࢆࢺࣝ ࢚ࣥ 2 mL ⁐ゎࡋࡓ㸬ࡑࡢࢺ࢚ࣝࣥ⁐ᾮ࣑ࣀᇶಟ 㣭࢝ࣂ࣮࢞ࣛࢫࢆᾐࡋ㸪 ࢩࣜࢥࣥ〇࢟ࣕࢵࣉ࡛ᐦ㛢ࡋ 4Υ ࡛෭༷ࡋࡓ㸬ḟ㸪ATRP 㛤ጞ BiBB 0.0465 g (0.202 mmol)ࡢ 247.5 μL ࢺ࢚ࣝࣥ⁐ᾮࢆㄪᩚࡋ㸪ࢹࢫ࣏࣮ࢧ ࣈࣝ⏝ࢩࣜࣥࢪࢆ⏝࠸࡚ࡇࡢ⁐ᾮࢆᚎࠎୗࡋ㸪30 min ෭༷ࢆ⾜ࡗࡓᚋ㸪25Υ㸪4 h ᛂࡋࡓ㸬ᛂ⤊ᚋ㸪 ࣓ࢱࣀ࣮ࣝ࠾ࡼࡧࢺ࢚ࣝࣥᾐࡋ㸪ᐊ ࡛ 15 min ㉸㡢Ἴ Ὑίࡋࡓ㸬࢞ࣛࢫ〇ࢫࢡ࣮ࣜࣗ⟶ AAm 0.119g (1.67 mmol), AN 0.018 g (0.332 mmol), CuBr(Ϩ) 0.010 g (0.0697 mmol), PMDETA 0.121 g (0.697 mmol), L-ࢫ ࢥࣝࣅࣥ㓟 0.123 g (0.697 mml)ࢆຍ࠼ DMSO 2 mL ⁐ゎࡋࡓ㸬ࡑࡢ DMSO ⁐ᾮ BiBB ࢆᅛᐃࡋࡓ࢝ࣂ࣮ ࢞ࣛࢫࢆᾐࡋ㸪40Υ㸪6 h 㔜ྜࢆ⾜ࡗࡓ㸬ᛂ⤊ᚋ㸪 － 56 － ࣓ࢱࣀ࣮ࣝ࠾ࡼࡧỈ࡛㉸㡢ἼὙίࢆ⾜࠸㸪┿✵⇱ࡋ㸪 poly(AAm-co-AN)-g-glass ࢆᚓࡓ㸬 JJODVV ⾲㠃ࡢ࢟ࣕࣛࢡࢱࣜࢮ࣮ࢩࣙࣥ Ỉᑐࡍࡿ᥋ゐゅࡣ ᗘㄪ⠇ࢫࢸ࣮ࢪࡁࡢ Erma ග Ꮫ♫〇ࢦࢽ࣓࣮࢜ࢱ࣮ᘧ᥋ゐゅ ᐃᶵ G-1 ᆺࢆ⏝࠸࡚ ᐃࡋࡓ(20)㸬ࡇࡢࡁ㸪⇕㟁ᑐࢆࢫࢸ࣮ࢪୖ᥋ゐࡉࡏ⾲ 㠃 ᗘࢆィ ࡋ࡞ࡀࡽ±0.1Υ࡛ ᗘㄪ⠇ࡋࡓ㸬ࢫࢸ࣮ࢪ ᗘࢆ 26Υࡋࡓᚋ㸪ᇶᯈୖ 3 μL ࡢỈࢆ⨨࠸ࡓ㸬1 min ௨ෆỈࡢᕥྑࡢ᥋ゐゅࢆ ᐃࡋࡓ㸬 ᐃࡣィ 5 ᅇࡢ࠺ࡕ㸪್᭱࠾ࡼࡧ᭱ᑠ್ࢆ㝖࠸ࡓᖹᆒࢆ᥋ゐゅ ࡋࡓ㸬 ࡲࡓ ᐃ ᗘࢆ40Υୖ᪼ࡉࡏྠᵝࡢ᧯సࢆ⾜࠸㸪 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┦ ᳨ฟࢩࢫࢸ࣒ࡼࡿ⾲㠃ࡢ㧗ప ᐃࡽホ౯ࡋࡓ㸬 +H/D ⣽⬊ࡢ᥋╔ቑṪ ㄪ〇ࡋࡓ g-glass ࡣගᏛࣇ࣒ࣝᅛᐃ⏝㏱᫂୧㠃ࢸ࣮ ࣉ HJ-3160W㸦᪥ᮾ㟁ᕤ㸦ᰴ㸧ࢆࢢࣛࣇࢺ㠃ࡢ㠃㈞ ࡾࡅ㸪g-glass ࢆ 4 ࢙࢘ࣝࡢ࣐ࣝࢳࢹࢩࣗ㸦࢙࢘ࣝࢧ ࢬ φ15 mm㸸PS㸧(Nunc)ᅛᐃࡋࡓ㸬ࢡ࣮ࣜࣥ࣋ࣥ ࢳ୰࡛ᅛᐃࡋࡓ g-glass ⾲㠃ࢆ࣮࢜ࢺࢡ࣮ࣞࣈ⁛⳦῭ࡢ ⁛⳦Ỉ࡚ 1 mL ࡛ 3 ᅇὙίࡋࡓ㸬ࡑࡢᚋ㸪ࢡ࣮ࣜࣥ࣋ ࣥࢳ୰ࡢ UV ⁛⳦ⅉୗ⣙ 30 cm ࡢ㊥㞳࣐ࣝࢳࢹࢩࣗ ࡢࢆ㛤ࡅ࡚㟼⨨㸪g-glass ⾲㠃ࢆ 1 h UV ↷ᑕࡋ࡚⁛⳦ ࡋࡓ㸬HeLa ⣽⬊ࢆ 10㸣FCS㸪1㸣NEAA ྵ᭷ MEM ᇵ ᆅ࡛ᇵ㣴ࡋࡓ㸬ࡑࡢᚋ㸪HeLa ⣽⬊ࡣ 1 mL ࡢ Trypsin ⁐ᾮ(2.5 mg mL-1)ࢆῧຍࡋ࡚ࡣࡃ㞳ࡋࡓ㸬HeLa ⣽⬊ࡣ ᾋ㐟⣽⬊⃰ᗘࢆ(3.0×104 cells mL-1)ㄪ〇ᚋ㸪800 μL ࢆ ୖグ g-glass ୖࢆᅛᐃࡋࡓ࢙࢘ࣝୖຍ࠼㸪ࣥ࢟ࣗ࣋ ࣮ࢱ࣮㸦APC-50D㸸ࢫࢸࢵࢡ㸧5%㓟Ⅳ⣲㞺ᅖẼ ୗ 30Υ㸪24 h ᇵ㣴ࡋࡓ㸬 +H/D ⣽⬊ࡢ ᗘ่⃭ࡼࡿࡣࡃ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❧ᆺࣜࢧ࣮ࢳ㢧ᚤ㙾 (IX71 㸸OLYMPUS) ࡛ほᐹࡋ㸪㢧ᚤ㙾ࢹࢪࢱ࣓ࣝ࢝ࣛ (DP72-BSW)ࢆ⏝࠸࡚ᙳࡋࡓ㸬ࡑࡢᚋ㸪 ᗘ่⃭ࡼ ࡿࡣࡃ㞳ࢆ⾜࠺ࡓࡵ㸪 ᗘࡀ 37Υタᐃࡢ࣮ࣥ࢟ࣗ࣋ࢱ ࣮㸦SCA-165D㸸ࢫࢸࢵࢡ㸧 g-glass ࢆᅛᐃࡋ࡚࠸ࡿ ࣐ࣝࢳࢹࢩࣗࢆ⛣ࡋ 30 min 㟼⨨ࡋࡓ㸬 㟼⨨ᚋ㸪800 μL ࡢ PBS ࢆ⏝࠸㸪3 ᅇὙίࡋ࡚ࡣࡃ㞳⣽⬊ࢆ㝖ཤࡋ㸪࢞ࣛ ࢫୖṧᏑࡍࡿ᥋╔⣽⬊ᩘࢆಽ❧ᆺࣜࢧ࣮ࢳ㢧ᚤ㙾 (IX71㸸OLYMPUS)࡛ほᐹࡋ㸪㢧ᚤ㙾ࢹࢪࢱ࣓ࣝ࢝ࣛ (DP72-BSW)ࢆ⏝࠸࡚ᙳࡋࡓ㸬⣽⬊ᩘࡢィ ࡣ㸪ಽ❧ ᆺࣜࢧ࣮ࢳ㢧ᚤ㙾(IX71㸸OLYMPUS)ࡢほᐹࡽぢ✚ࡶ ࡗࡓ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㸪ୖࡳࢆ ྲྀࡾ㝖ࡁ㸪ṧᏑࡍࡿ᥋╔⣽⬊ᩘࢆྠᵝィ⟬ࡋࡓ㸬ࡣࡃ㞳 ⋡(%)ࡣ᥋╔⣽⬊ᩘࡢᕪࡽ⟬ฟࡋࡓ㸬 ⤖ᯝ⪃ᐹ 3RO\$$PFR$1ࢢࣛࣇࢺ࢞ࣛࢫࡢㄪ〇⾲㠃࢟ࣕ ࣛࢡࢱࣜࢮ࣮ࣥࣙࣥ ⣽⬊ᇵ㣴࠾ࡼࡧࡣࡃ㞳ᇶᮦࡋ࡚ࡢᛂ⏝ࡉࡽࡣ CW ࣮ࣞࢨ࣮↷ᑕᶵࢆ⏝࠸ࡓᒁᡤⓗ࡞ຍ ࡼࡿ⣽⬊ࡢ㑅ᢥ ⓗ ࡞ ࡣ ࡃ 㞳 ࣭ ᅇ ࢆ ┠ ⓗ ࡋ 㸪 UCST ࢆ ♧ ࡍ poly(AAm-co-AN)-g-glass ࡢㄪ〇ࢆ⾲㠃㛤ጞཎᏊ⛣ືࣛ ࢪ࢝ࣝ㔜ྜἲࡼࡾヨࡳࡓ㸬⾲㠃㛤ጞ㔜ྜᚋࡢ࢞ࣛࢫ⾲ 㠃ࡢ poly(AAm-co-AN)ࡢಟ㣭ࢆ☜ㄆࡍࡿࡓࡵ㸪Ẽ㞺ᅖ Ẽୗ࡛ࡢ⾲㠃࡛ࡢ SPM ᐃ(30 μm×30 μm)ࡢ⤖ᯝࢆ Fig.1 ♧ࡍ㸬ᮍฎ⌮࢞ࣛࢫ࡛ࡣพฝࡀ↓ࡃࣇࣛࢵࢺ࡛࠶ ࡿࡇᑐࡋ㸪 poly(AAm-co-AN)ࢆࢢࣛࣇࢺ㔜ྜࡋࡓ g-glass ࡛ࡣࡑࢀࡒࢀ㸪ࡇࢀࡽ࣏࣐࣮ࣜ⏤᮶⪃࠼ࡽࢀ ࡿࢼࣀࢫࢣ࣮࡛ࣝࡢࣈࣛࢩ≧ࡢพฝࡀ⾲㠃ୖほᐹࡉࢀ ࡓ㸬ḟ㸪 g-glass ࡢỈᑐࡍࡿ᥋ゐゅ(θ)ࢆ ᐃࡋࡓ⤖ Fig.1 SPM (b)g-glass. image of glasses; (a)Untreated, ᯝ㸪ࢫࢸ࣮ࢪ ᗘ 26Υ࡛ࡣᮍฎ⌮࢞ࣛࢫࡢ θ=21rᑐ ࡋ࡚ poly(AAm-co-AN)ࢆಟ㣭ࡋࡓ g-glass ࡛ࡣ θ=69r ࡞ࡾ poly(AAm-co-AN)ᅛᐃࡼࡾ θ ࡀቑࡋ㸪࢞ࣛࢫ ⾲㠃ࡀỈᛶኚࡋࡓࡇࢆㄆࡵࡓ㸬୍᪉㸪ࢫࢸ࣮ࢪ ᗘࢆ 40Υୖࡆ ᐃࡋࡓ θ ࡣᮍฎ⌮࢞ࣛࢫࡣኚࡋ࡞ ࠸ࡢᑐࡋ㸪g-glass ࡛ࡣ θ=65r ᗘୖ᪼ࡼࡗ࡚㸪 ⾲㠃ࡢࢀᛶࡀቑࡋ࡚ぶỈᛶኚࡋࡓ㸬ࡇࢀࡣ㸪࢞ ࣛࢫ⾲㠃ࢢࣛࣇࢺࡋࡓ poly(AAm-co-AN)ࡀ UCST ௨ ୖ࡞ࡾ㸪࣏࣐࣮ࣜ㙐ࡀࢢࣟࣅ࣮ࣗࣝ≧ࡽࣛࣥࢲ࣒ࢥ ࣝ≧ኚࡋ㸪⾲㠃ࡢࢀᛶࡀỈᛶࡽぶỈᛶ ኚࡋࡓࡓࡵ⪃࠼ࡽࢀࡿ㸬 － 57 － ࣐࣮ࣜࡀ࣏࣐࣮ࣜ㙐㛗ཬࡧ፹యࡢሷ➼ࡁࡃᙳ㡪ࢆཷ JJODVV ⾲㠃࡛ࡢ⣽⬊ᇵ㣴 ᗘ่⃭ࡼࡿࡣࡃ㞳 3.1 ⠇࡛ ᗘ่⃭ࡼࡾぶ࣭Ỉᛶࡀኚࡋ㸪SPM ᐃࡼࡾ⾲㠃ᛶ≧ࡢኚࢆㄆࡵࡓ g-glass ୖࡢ⣽⬊ᇵ㣴 ࡞ࡽࡧຍ 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 ࢙࢘ࣝࡢᇵ㣴ࢩ ࣮ࣕࣞ┤ᚄ 12 mm ࡢ g-glass ࠾ࡼࡧᮍฎ⌮࢞ࣛࢫࢆ࢘ ࢙ࣝୖ㏱᫂୧㠃ࢸ࣮ࣉ࡛㈞ࡾࡅࡓ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ᅗࡽ 30Υ࡛ HeLa A-1 100 90 % of exfoliation 80 70 60 50 He L a cells 40 30 20 10 A-2 0 Untreated Untreated B-1 g-glass Fig.3 Mechanical and thermal stimuli-exfoliation of HeLa cells attached on g-glass at 37 Υ after cultivation at 30Υ for 24 hr. B-2 g-glass Fig.2 Phase-contrast microscope observation of HeLa cells attached glsses; (A)30°C and (B) 37°C. ⣽⬊ࡣ g-glass ୖ࢞ࣛࢫᇶᯈୖྠᵝ᥋╔࣭ఙᒎࡋቑ Ṫྍ⬟࡛࠶ࡗࡓ㸬ḟ㸪ຍ ࡼࡾ⾲㠃ࡀỈᛶࡽぶ Ỉᛶࡢᛂ⟅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 ᇶᯈࡢᮍ᥋╔ࡢ⣽⬊ࢆྲྀࡾ㝖ࡁ㸪37Υࡢ࣮ࣥ࢟ࣗ࣋ ࢱ࣮ g-glass ࢆ᥋╔ᅛᐃࡋ࡚࠸ࡿ 4 ࢙࣐࢘ࣝࣝࢳࢹ ࢵࢩࣗࢆ⛣ࡋ㸪30 min 㟼⨨ࡋࡓ㸬ḟ㸪PBS(25Υ)࡛ 3 ᅇὙίࡋ㸪ࡣࡃ㞳ࢆ⾜ࡗࡓ㸬ຍ ࡼࡿ HeLa ⣽⬊ࡢࡣ ࡃ㞳⋡ࢆᮍฎ⌮࢞ࣛࢫేࡏ࡚㸪᥋╔ṧᏑࡍࡿ⣽⬊ᩘ ࡽぢ✚ࡶࡗࡓࡣࡃ㞳⣽⬊ᩘࡢྜࢆ Fig.3 ♧ࡍ㸬 g-glass ࡣࡣࡃ㞳⋡⣙ 98㸣ᮍฎ⌮࢞ࣛࢫ 㸦ࡣࡃ㞳⋡ 9%㸧 ࡼࡾࡶ 90%⛬ᗘୖ᪼ࡋࡓ㸬ࡇࢀࡼࡾ㸪UCST ࢆ♧ࡍ poly(AAm-co-AN)ࢢࣛࣇࢺ㙐ࡢᅛᐃࡼࡾ㸪UCST ௨ ୗࡢ ᗘ࡛᥋╔ࡋࡓ HeLa ⣽⬊ࡣ UCST ௨ୖຍ ࡍࡿ ࡇࡼࡾࡣࡃ㞳ࢆಁ㐍ࡍࡿຠᯝࢆㄆࡵࡓ㸬௨๓◊✲ࢆ ⾜ࡗ࡚࠸ࡓ UCST ࢆ♧ࡍ୧ᛶ࢜ࣥᆺ㸦2-࣓ࢱࢡࣜࣟ ࣝ࢜࢟ࢩ㸧࢚ࢳࣝࢪ࣓ࢳࣝ-3-ࢫࣝ࣍ࣉࣟࣆࣝࣥࣔࢽ࢘ ࣒ࣄࢻࣟ࢟ࢧࢻ(SBMA)(21),(22)㙐ࢆᅛᐃࡋࡓ࢞ࣛࢫᇶ ᯈࢆ⏝࠸ࡓྠᵝࡢᐇ㦂࡛ࡣ UCST ࡣ♧ࡍࡀ⣽⬊ࢆ⏝࠸ࡓ ᗘ่⃭ࡣࡃ㞳࡛ࡣᮍฎ⌮࢞ࣛࢫẚⓗ࡞ࡣࡃ㞳⋡ ྥୖࡣㄆࡵࡽࢀ࡞ࡗࡓ㸬ࡇࢀࡣ୧ᛶ࢜ࣥᆺ UCST ࣏ ࡅࡿ⪃࠼ࡽࢀ࡚࠸ࡿࡓࡵ࡛࠶ࡿ㸬ࡑࡢࡓࡵ㸪ሷࡀྵࡲ ࢀ࡚࠸ࡿ PBS ୰࡛ࡣᇶᯈୖᅛᐃࡉࢀࡓ࣏࣐࣮ࣜࡀ ሷࡼࡾ┦㌿⛣ᣲືࡢᙳ㡪ࢆཷࡅࡓࡓࡵ UCST ᶵ⬟ࢆⓎ ࡛ࡁ࡞ࡗࡓ⪃࠼ࡽࢀࡿ㸬ᅇ⏝ࡋࡓࣀࢽ࢜ࣥᆺ UCST ࣏࣐࣮ࣜࡣࡑࡢࡼ࠺࡞ᙳ㡪ࢆཷࡅࡿࡇࡀ࡞࠸ࡓ ࡵ㸪ࡣࡃ㞳⋡ࡢ㣕㌍ⓗ࡞ྥୖࡘ࡞ࡀࡗࡓ⪃࠼ࡽࢀࡿ㸬 ࡲࡓ㸪 ᗘᛂ⟅ᛶ࣏࣐࣮ࣜ㙐ᅛᐃ⾲㠃ࡣ࣏࣐࣮ࣜ㙐ࡢ ᐦᗘࡸศᏊ㔞ࡁࡃᙳ㡪ࢆཷࡅࡿࡇࡽ(23) 㸪⣽⬊✀ ࡢ㐪࠸ࡼࡿ⣽⬊᥋╔ཬࡧࡣࡃ㞳᭱㐺࡞ᛶ≧ᅛᐃ ࣏࣐࣮ࣜ㙐ࢆᚋㄪᩚࡍࡿᚲせࡀ࠶ࡿ⪃࠼ࡽࢀࡿ㸬 ࡲࡵ ձSPM ᐃ࠾ࡼࡧỈࡢ᥋ゐゅࡢ ᐃ⤖ᯝࡼࡾ㸪⾲㠃 㛤ጞཎᏊ⛣ືࣛࢪ࢝ࣝ㔜ྜἲࡼࡗ࡚࢞ࣛࢫᇶᯈ⾲㠃 poly(AAm-co-AN)ࡢᅛᐃࢆㄆࡵࡓ㸬 ղpoly(AAm-co-AN)ࢆᅛᐃࡋࡓ࢞ࣛࢫࡣ 26Υࡽ 40Υຍ ࡼࡾ᥋ゐゅࡀపୗࡋ⾲㠃ࡀỈᛶࡽぶỈ ᛶኚࡍࡿ UCST ᛶࢆ♧ࡋࡓ㸬 ճ HeLa ⣽ ⬊ ࢆ ⏝ ࠸ ࡓ ᥋ ╔ ࣭ ࡣ ࡃ 㞳 ヨ 㦂 ࡛ ࡣ 㸪 poly(AAm-co-AN)ಟ㣭࢞ࣛࢫᇶᯈࡣ㸪30Υ࡛ࡣ⣽⬊ࡀ࢞ ࣛࢫᇶᯈ⾲㠃᥋╔࣭ቑṪࡋࡓ㸬poly(AAm-co-AN)ᅛᐃ ࢞ࣛࢫࡣ37Υ ᗘࢆୖ᪼ࡍࡿࡇࡼࡿ⣽⬊ࡢࡣࡃ 㞳⋡ࡢྥୖࢆㄆࡵࡓ㸬 ௨ୖࡢ⤖ᯝࡽ㸪UCST ࢆ♧ࡍࣀࢽ࢜ࣥᆺ࣏࣐࣮ࣜࡢ ᅛᐃࡼࡾ㸪ຍ ࡼࡿ ᗘ่⃭ࡼࡾ⣽⬊ࡣࡃ㞳ࡀ ྍ⬟࡛࠶ࡿ⪃࠼ࡽࢀࡿ㸬᥋╔⣽⬊ࡢࡣࡃ㞳⋡ࡢࡉࡽ࡞ ࡿྥୖࡢࡓࡵࡣ㸪ᅛᐃ࣏࣐࣮ࣜࡢ㙐㛗㸪ᐦᗘ㸪⤌ᡂ － 58 － ➼ࡉࡽࡣࡣࡃ㞳ࡢ ᗘ➼ࡢ᭱㐺ࡀᚲせ࡛࠶ࡿ㸬 ຍ ࡼࡿࡣࡃ㞳ᛶ⬟ࡢ☜❧ࡼࡗ࡚㸪UCST ᆺ࣏ࣜ ࣐࣮ᅛᐃ ǍAy CW ࣮ࣞࢨ࣮↷ᑕᶵࢆ⏝࠸ࡓࢩࢫࢸ࣒ ࡼࡗ࡚┠ⓗ⣽⬊ࡢ㑅ᢥⓗ࡞ࡣࡃ㞳࣭ᅇࢶ࣮ࣝࡢస〇 ᛂ⏝ࡀ࡛ࡁࡿ⪃࠼ࡽࢀࡿ㸬 ㅰ㎡ ᮏ◊✲ࡢ୍㒊ࡣ⤒῭⏘ᴗ┬ᆅᇦࣀ࣮࣋ࢩࣙࣥฟ㛤 Ⓨᴗཬࡧᡓ␎ⓗᇶ┙ᢏ⾡㧗ᗘ᥎㐍ᴗࡢᨭࡼࡿ ࡶࡢ࡛࠶ࡾ㸪ࡇࡇㅰពࢆ⾲ࡋࡲࡍ㸬 (21)Ai T. 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