中國醫藥大學機構典藏 China Medical University Repository, Taiwan:Item 310903500/41364

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(1)ύ ୯ ᙴ ᛰ ε Ꮲ ୷ᘵᙴᏢࣴ‫܌ز‬ ᅺγᏢՏፕЎ n-Butylidenephthalide (BP)೸ၸࢲϯ Jak2-Stat3 ૻ৲ ໺ሀ೼৩ᆢ࡭༸ಒझ‫ޑ‬ӭૈ‫܄‬ n-Butylidenephthalide (BP) maintained the pluripotency of stem cells by activating Jak2-Stat3 signaling pathway. ࡰᏤ௲௤Ǻቅ၃ѳ ௲௤. ࣴ‫ز‬ғǺᙁみ⃐ ύ ๮ ҇ ୯ ΋ɄɄԃΎД.

(2) ύЎᄔा ᇨᏤࠠӭૈ༸ಒझ(Induced pluripotent stem cellsǴᙁᆀ iPS cells)ࢂᙖҗ ੰࢥ‫س‬಍ᙯࢉ 4 ঁᙯᒵӢη(Oct4, Sox2, c-Myc ‫ ک‬Klf4)຾Εᡏಒझ‫܌‬ౢғǶ ᗨฅҞ߻Ԗ೚ӭౢғ iPS cells ‫ޑ‬Б‫ݤ‬Ǵՠਏ౗եϝฅࢂ΋ঁࡐε‫ޑ‬ምᛖሡ ाլܺǶԜѦǴiPS ᆶखज༸ಒझ(embryonic stem cellsǴᙁᆀ ES cells)΋ኬ ሡाқՈౚቚғ‫ڋ׭‬Ӣη(Leukemia inhibitory factorǴᙁᆀ LIF)ٰᆢ࡭༸ಒ झ‫ޑ‬ӭૈ‫܄‬Ǵՠ LIF ሽ਱ܳ຦ǶҁጇፕЎයఈёа‫ډפ‬΋ঁவύ૛ᛰ๧‫ڗ‬ р‫ޑ‬પϯӝ‫ނ‬Ҕٰᆢ࡭ ES ‫ ک‬iPS cells ‫ޑ‬ӭૈ‫܄‬а‫ڗ‬ж LIFǴӕਔΨёаቚ у iPS cells ғԋ‫ޑ‬ਏ౗Ƕ‫ॺך‬ว౜྽λႵᠼᆢ҆ಒझ࿶ၸவ྽ᘜ(Angelica sinensis)๧‫ ޑڗ‬n-Butylidenephthalide (BP) 10g/ml ೀ౛ࡕǴёаቚу Oct4 ‫ ک‬Sox2 ‫୷ޑ‬Ӣ߄౜ໆǹΨว౜྽ BP ᐚࡋගଯ(20g/ml ‫ ک‬40g/ml)ਔǴϸ ԶբҔफ़եǶ‫ॺך‬ճҔ 3-[4,5-dimethylthiahiazo-2-y1]-2,4-diphenytetrazolium bromide (MTT assay)ٰෳ၂೭٤ᐚࡋࢂց཮೷ԋಒझ঒ΫǴԶኧᏵᡉҢຬၸ 80g/ml BP ω཮೷ԋಒझ঒ΫǶ‫ॺך‬Ψ٬Ҕόӕᐚࡋ‫ ޑ‬BP (‫୻ܭ‬Ꭶనύ‫ڗ‬ ж LIF)Ǵෳ၂ BP ࢂցёаԖਏ‫ޑ‬ᆢ࡭ ES Ϸ iPS cells ‫ޑ‬ӭૈ‫܄‬Ƕ࿶ၸ༸ಒ झ኱૶(stem cell markers)‫ޑ‬ෳ‫ۓ‬ϐࡕǴᡵ‫܄‬ᕗለ䁙(alkaline phosphatase)ǵ SSEA1 ‫ ک‬Nanog ‫߄ޑ‬౜ໆӧ BP ๏ϒ‫ޑ‬ჴᡍಔଯ‫ܭ‬όу LIF ‫ޑ‬௓‫ڋ‬ಔǹճ Ҕ embryoid body ‫׎ޑ‬ԋ‫ک‬ϩϯٰዴᇡ BP ёа٬ ES cells ᆢ࡭‫ځ‬ӭૈ‫܄‬Ƕ ᛾ჴ BP ёаԖਏ‫ޑ‬ᆢ࡭ ES Ϸ iPS cells ‫ޑ‬ӭૈ‫܄‬ǶԜѦǴ‫ॺך‬٬Ҕ microarray. II.

(3) analysis ٰ‫פ‬൨ BP Ьाፓ௓‫ૻޑ‬৲೼৩Ƕ่݀ᡉҢǴ‫ ڙ‬BP ፓ௓‫߻ޑ‬ΟӜ ૻ৲೼৩ϩձࣁǺPPAR, ECM ‫ ک‬Jak-Stat pathwayǶ‫ॺך‬ୀෳ‫ډ‬࿶ BP ೀ౛ ၸࡕǴphospholation-Jak2 ‫ ک‬phospholation-Stat3 ೈқ፦‫߄ޑ‬౜ໆ೿ԖቚуǴ ᆶ Jak2-Stat3 pathway ࣬ᜢ‫ޑ‬ಒझᐟન(Cytokines)୷Ӣ߄౜ໆΨԖቚу‫ޑ‬౜ ຝǶനࡕǴ‫ॺך‬٬Ҕ pou5f1-GFP MEF cells ٰෳ၂࿶җ BP ೀ౛ࡕ iPS cells ғԋ‫ޑ‬ਏ౗ǶኧᏵᡉҢǴBP ೀ౛ࡕᡉ๱ቚуౢғ GFP positive clones ‫ޑ‬ਏ ౗Ƕᕴٰ่ᇥǴ‫ޑॺך‬ኧᏵᡉҢǴBP ᙖҗᇨᏤಒझᐟન߄౜Ǵࢲϯ Jak2-Stat3 pathway ᆢ࡭ iPS cells ‫ޑ‬ӭૈ‫܄‬ǶԜѦǴBP ᗋёаගଯ iPS cells ғԋ‫ޑ‬ਏ ౗Ƕ. III.

(4) Abstract The first induced pluripotent stem (iPS) cells were generated in 2006 from somatic cells by introducing Oct4, Sox2, c-Myc and Klf4. The original process was inefficient, and maintaining the pluripotency of embryonic stem (ES) and iPS cell cultures required leukemia induced factor (LIF) as an expensive reagent. Our goal is to find a pure compound that not only maintains ES and iPS cell pluripotency, but also increases iPS cell generation efficiency. From 15 candidate compounds we determined that 10 g/ml n-Butylidenephthalide (BP), an Angelica sinensis extract, triggers up-regulation of Oct4 and Sox2 gene expression levels in MEF cells. We used ES and iPS cells treated with different concentrations of BP to test its usefulness for maintaining stem cell pluripotency. Results indicate higher expression levels of several stem cell markers in BP-treated ES and iPS cells compared to controls that did not contain LIF including alkaline phosphatase, SSEA1, and Nanog. Embryoid body formation and differentiation results confirm that BP containing medium culture is capable of maintaining ES cell pluripotency. Microarray analysis data identified PPAR, ECM and Jak-Stat signaling as the top three deregulated pathways. We subsequently determined that phospholation-Jak2 and phospholation-Stat3 protein levels increased following BP treatment, and that cytokines associated with the Jak2-Stat3 pathway were up-regulated. Last, we used pou5f1-GFP MEF IV.

(5) cells to test iPS generation efficiency following BP treatment. Our data demonstrate the ability of BP to maintain stem cell pluripotency via the Jak2-Stat3 pathway by inducing cytokine expression levels, at the same time improving iPS generation efficiency.. V.

(6) ठᖴ! ስ୉޸໒‫ۑޑ‬࿯ǴསҢ๱ᅺγ੤‫ٿ‬ԃ‫ޑ‬ғࢲςௗ߈‫׀‬ᖂǴΨж߄‫ޑך‬Ꮲ ғғఱջஒฝ΢ѡᗺǴ‫ٿ‬ԃٰ‫ޑ‬ჴᡍ࠻ғࢲԖӵΟྕཪ૓Ǵ࿶ᐕၸեዊǵ ਋‫ש‬ǵᑫᏟයࡑᗋԖᅐߏ‫ޑ‬฻ࡑǴಖ‫ز‬໩ճ‫ډ‬ၲಖᗺǶӧ೭΋ၡ΢Ǵߚத གᖴ೚ӭ‫ޑ‬ΓᔅշᆶගᗺǴ٬‫ך‬ӧᏢೌ‫ޕ‬᛽‫ঁک‬Γว৖΢ᕇளᙦ൤‫ޑ‬кჴ ᆶԋߏǶനᡣ‫ך‬གᖴ‫ޑ‬ΓǴ྽ฅࢂ‫ࡰޑך‬Ꮴ௲௤ቅ၃ѳԴৣǴ‫ٿ‬ԃٰ‫ࡰޑ‬ Ꮴǵ࿎ߦ‫ک‬х৒Ǵ‫ך‬ωёа໩ճ೯ၸ೭ख़ख़‫ޑ‬ԵᡍǶགᖴම࿶ᔅշϷ௲Ꮴ ၸ‫ޑך‬ԴৣॺǴாॺ‫ޑ‬ගᗺࢂ‫ך‬ӧԾ‫ػ୻ך‬ၸำύ‫ޑ‬΋࿈ܴᐩǶགᖴჴᡍ ࠻‫܌ޑ‬Ԗշ౛Ꮲ‫ॺۆ‬Ǵӧჴᡍ΢፾ਔ‫ޑ‬ගᗺᆶᔅշǶགᖴჴᡍ࠻‫ޑ‬უՔॺǴ εৎό໻ӧჴᡍፐ཰΢ϕ࣬૸ፕǵϕ࣬ϪዲǴӧВதғࢲύΨಕᑈΑ‫ך‬΋ ԃӭٰ೚ӭ‫៿ޑ‬ઢᆶག୏Ƕགᖴ‫ך‬നᒃང‫϶࠻ޑ‬Ǵ೭٤ԃٰ‫ޑ‬ഉՔǴഉ‫ך‬ ϩ٦៿኷Ǵ᠋‫ך‬ว‫آ‬ផǶ࣬྽གᖴထထᆶထ༰೭‫ٿ‬ԃٰ‫៝ྣޑ‬ǶനࡕǴ྽ ฅࢂाགᖴ‫ך‬നᒃང‫ޑ‬ৎΓǴᖴᖴ‫ݿ‬༰ӄΚ‫ޑ‬Ѝ࡭ᆶधЈ‫ޑ‬ਭ୻ǴᗋԖঢ ঢॺ‫ޑ‬х৒Ǵ‫׆‬ఈ‫ޑך‬ոΚૈᡣாॺག‫ډ‬ᠠ໹ᆶ‫ݒ‬ኃǶ!. VI.

(7) Ҟᒵ! ύЎᄔा................................................................................................................... II Abstract .................................................................................................................. IV ಃ΋ക ߻‫ق‬..............................................................................................................1 1.1 ࣴ‫ز‬ङඳ.............................................................................................................1 1.1.1 iPS cells ‫ޑ‬ख़ा‫܄‬..................................................................................................1 1.1.2 Leukemia Inhibitory Factor (LIF) ‫ ک‬Jak2-Stat3 signaling pathway..........2 1.1.3 n-Butylidenephthalide (BP) ࣬ᜢғ‫܄ࢲނ‬ϐࣴ‫ ز‬.........................................4 1.2 ࣴ‫ز‬Ҟ‫ޑ‬....................................................................................................................4. ಃΒക ‫׷‬਑ᆶБ‫ ݤ‬.................................................................................................6 2.1 ಒझ୻Ꭶ.............................................................................................................6 2.1.1 ԴႵखजᠼᆢ҆ಒझ (Mouse Embryoic Fibroblasts)...................................6 2.1.2 iPS ‫ ک‬mES cells.....................................................................................................6. 2.2 ಒझӸࢲෳ၂ (MTT Assay)...........................................................................7 2.3 RNA ‫ޑ‬๧‫ڗ‬Ϸ cDNA ‫ޑ‬ᇙഢ..........................................................................8 2.4 ջਔᆫӝ䁙ೱᙹϸᔈ (Real-time Polymerase Chain Reaction)...............8 2.5 ᡵ‫܄‬ᕗለ䁙ࢉՅ (Alkaline Phosphatase Staining) .....................................9 2.6 խࣝᑻӀࢉՅ (Immunofluence stain)........................................................10 2.7 खजౚ‫׎ޑ‬ԋ‫ک‬ϩϯ(Embryoid body (EB) formation and differentiation)........................................................................................................10 2.8 ಒझྋှ‫ޑނ‬ᇙഢ..........................................................................................11 2.9 ౷ለΜΒ✊໊—ᆫЧ౎ㄽữᏉጤᡏႝ‫ݚ‬ȐSDS-PAGEȑ.......................12 2.10 ՋБᏀᗺ‫ݤ‬ȐWestern Blotting Analysisȑ..............................................13. VII.

(8) 2.11 DNA ༾ତӈϩ‫(݋‬DNA Microarray Analysis) .........................................14 2.12 iPS cells ғԋਏ౗‫ޑ‬ෳ‫ ۓ‬.............................................................................15 2.13 ಍ीϩ‫݋‬.........................................................................................................15 ಃΟക ࣴ‫ ่݀ز‬...................................................................................................16 3.1 ύ૛ᛰ‫ޑ‬ᑔᒧ ...................................................................................................16 3.2 BP ჹ MEF cells ғߏ‫ޑ‬ቹៜ.........................................................................16 3.3 BP ჹ Oct4ǵSox2ǵc-Myc ‫ ک‬Klf4 ୷Ӣ߄౜ໆ‫ޑ‬ቹៜ...........................16 3.4 BP ჹ ES ‫ ک‬iPS cells ᆢ࡭Ծ‫׳ך‬ཥૈΚ‫ޑ‬ቹៜ .......................................17 3.5 BP ჹ MEF cells ϩηૻ৲໺ሀ೼৩‫ޑ‬ቹៜ................................................19 3.6 BP ჹ Jak2 ‫ ک‬Stat3 ୷Ӣ߄౜ϐቹៜ .........................................................20 3.7 BP ჹ Jak2 ‫ ک‬Stat3 ೈқ፦߄౜ϐቹៜ.....................................................20 3.8 BP ჹ cytokine ‫୷ޑ‬Ӣ߄౜ϐቹៜ ...............................................................20 3.9 BP ჹ iPS cells ғԋϐቹៜ ............................................................................21 ಃѤക ૸ፕ............................................................................................................22 ୖԵЎ᝘..................................................................................................................27 კ߄...........................................................................................................................32. VIII.

(9) ಃ΋ക ߻‫ق‬ 1.1 ࣴ‫ز‬ङඳ 1.1.1 iPS cells ‫ޑ‬ख़ा‫܄‬ खज༸ಒझ(Embryonic stem celltǴᙁᆀ ES cells)ǴٰԾ‫ܭ‬থ٢୏‫៶ނ‬ख ϣ‫ޑ‬ϣखቫಒझǴ‫ڀ‬Ԗคज़ғߏૈΚ٠ЪёаϩϯࣁҺՖΟखቫύ‫ޑ‬ಒझ (1-3)Ƕҗ‫ܭ‬खज༸ಒझӕਔ‫ڀ‬ԖԾ‫׳ך‬ཥ‫ک‬ϩϯԋӭᅿಒझᜪࠠ‫ૈޑ‬ΚǴ ჹ‫ܭ‬ᖏ‫ޑ׉‬ಒझᕍ‫ݤ‬ǴᏱԖѮε‫ޑ‬ወΚ(4)ǶӢԜǴΓॺයࡑखज༸ಒझё аҔ‫ܭ‬Αှ҂‫ੰޑޕ‬౛ᐒᙯǴཥᛰ໒วύᑔᒧԖਏ‫ک܄‬Ӽӄ‫ࡰޑ܄‬኱Ǵа Ϸ‫ݯ‬ᕍ‫ک‬௓‫ڋ‬Ӛᅿ੯ੰ(3, 5)Ǵ‫ٯ‬ӵǺମᡎ౽෌[hematopoietic stem cells (HSCs)]ᔈҔ‫ܭ‬қՈੰǵՈ϶ੰ‫ک‬೦Ոǹ໔፦༸ಒझ[Mesenchymal stem cells (MSCs)]ᔈҔ‫ܭ‬લՈ‫܈‬ཞ໾‫ک‬຾ՉԖਏ‫ޑ‬ՈᆅӆғǴঅൺ‫ڙ‬ཞ୔ୱ(6)Ƕӧа ߻‫زࣴޑ‬ύǴखज༸ಒझёаϩϯԋӭЃữઓ࿶ϡ຾Չಒझ‫ݯ‬ᕍ(7)ǶՠࢂǴ खज༸ಒझҔ‫ܭ‬ಒझ‫ݯ‬ᕍԖ‫ঁٿ‬εୢᚒǺ΋Бय़Ѹሡय़ᖏউ౛ၰቺ‫ݾ‬᝼‫ޑ‬ ୢᚒǴќ΋Бय़߾ࢂ஻‫ޣ‬౽෌ࡕౢғխࣝ௨Ѿ‫ޑ‬ୢᚒǴᏤठखज༸ಒझ‫ޑ‬ ᔈҔ‫ߔډڙ‬ᛖǶ ᇨᏤࠠӭૈ༸ಒझ(Induced pluripotent stem cellsǴᙁᆀ iPS cells) ࢂࣴ‫ز‬ ༸ಒझ‫ݯ‬ᕍ࣬྽ख़ा‫ޑ‬ཥᑉሦୱǶiPS cells ࢂஒ Oct4, Sox2, c-Myc ‫ ک‬Klf4 ೭Ѥঁ୷Ӣᙯࢉ຾ΕԴႵ‫܈‬Γᜪ‫ޑ‬ᠼᆢ҆ಒझ‫܌‬ғԋ(3, 8)ǶiPS ᆶ ES cells ‫ޑ‬੝‫ߚ܄‬த࣬՟Ǵό໻ࢂቚ෗ૈΚǵಒझࠠᄊǵ୷Ӣ߄౜ǵಒझ߄य़‫ל‬চǵ. 1.

(10) ᆄಈ䁙ࢲ‫܄‬ǴᗋԖ୷Ӣ΢ፓ௓Ҙ୷ϯ‫ ޑ‬AP genetic site ೿࣬྽΋ठǶiPS cells ‫ڀ‬Ԗ‫ޑ‬ӭૈ‫܄‬ჹ‫ܭ‬ಔᙃঅൺ‫׳ک‬ඤࢂ΋໨зΓᑫᏟ‫ޑ‬ወΚ(9)Ƕ‫׳‬ख़ा‫ޑ‬ ࢂǴiPS cells ό໻ёаᗉ໒ၰቺ‫ޑ‬ୢᚒǴΨёаᗉ໒խࣝ௨ѾǶՠࢂǴ౜໘ ࢤ iPS cells ‫ޑ‬ᔈҔय़ᖏനεᜤᚒࢂ iPS cells ‫ޑ‬ౢғਏ౗ߚதեǴቚуᔈҔ ӧੰΓ‫ي‬΢‫֚ޑ‬ᜤ(10)ǶҞ߻ǴiPS cells ‫ےزࣴޑ‬ԑࢂा‫ډפ‬΋ঁᙁൂԶЪ Ӽӄ‫ޑ‬Б‫ݤ‬ёаଯਏ౗‫ޑ‬ౢғ iPS cellsǴ٬ iPS cells ‫ޑ‬ᔈҔ‫׳‬уቶ‫ݱ‬Ƕ 1.1.2 Leukemia Inhibitory Factor (LIF) ‫ ک‬Jak2-Stat3 signaling pathway ᗨฅٰԾόӕ୏‫ޑނ‬खज༸ಒझ੝‫ک܄‬ᆢ࡭཮Ԗ‫܌‬όӕǴԴႵ‫ޑ‬खज༸ ಒझ୻ᎦਔǴሡा೸ၸқՈౚቚғ‫ڋ׭‬Ӣη(Leukemia inhibitory factorǴᙁ ᆀ LIF)ٰᆢ࡭खज༸ಒझ‫ޑ‬Ծ‫׳ך‬ཥૈΚ(11-13)ǶLIF ࢂឦ‫ ܭ‬IL-6 ৎ௼‫ޑ‬ ΋঩ǴѬёаᇨᏤԴႵखज༸ಒझғߏǴ٠Ъ‫ڋ׭‬ԴႵखज༸ಒझϩϯ (14-16)ǶLIF ߾ࢂ೸ၸᆶ LIF receptor ‫ ک‬gp130 [IL-6 ৎ௼‫ޑ‬Ӆӕ‫ڙ‬ᡏ(IL-6, IL-11, CNTF, OSM ‫ ک‬LIF)] (17-21)ፄӝ‫่ނ‬ӝǴࢲϯ Janus kinase (Jak)‫ک‬ signal transducer and activator of transcription (Stat)ǴԶ٬खज༸ಒझёаᆢ࡭ Ծ‫׳ך‬ཥૈΚ‫ک‬ӭૈ‫܄‬Ȑკ΋ȑ(22-24)Ƕ Jak (Janus Kinases)‫ޑ‬ৎ௼ԋ঩х֖ Jak1ǵJak2ǵJak3ǵTYK2 (Tyrosine Kinase 2)ǴѬॺՏ‫ܭ‬ಒझጢϣୁ٠ߕ๱‫ڙܭ‬Ꮤ΢Ǵࣣឦ‫ڙߚܭ‬Ꮤೈқᐟ䁙 (nonreceptor protein kinases)Ǵϩηໆϟ‫ ܭ‬120~135 KDa ϐ໔ǹJaks ֖Ύঁᐟ 䁙୔ୱ(JH domains 1~7)ǴՠѝԖ C ᆄ‫ ޑ‬JH1 domain ‫ڀ‬ഢ໽ϯࢲ‫܄‬ǴԶ N. 2.

(11) ᆄϐ JH6 domain ‫ ک‬JH7 domain ᆶ‫ڙ‬Ꮤϐ໔ࣣԖҬϕբҔ(25)Ƕ ӧ Jak ‫ޑ‬ৎ௼ԋ঩ύǴJak2 ‫ܭ‬ҁጇፕЎ္‫ת‬ᄽ΋ঁख़ा‫فޑ‬ՅǹJak2 ࢂୖᆶ‫ ܭ‬cytokine receptor signaling ύႀữለᐟ䁙ৎ௼‫ޑ‬΋঩Ǵᙖҗ IL-6 ৎ ௼‫ ޑ‬cytokines ೸ၸ gp130 ‫ځ܈‬дଯ߄౜‫ ޑ‬LIFR ٰӅӕࢲϯ Jak2 (26-29)Ǵ ຾Զ٬ Stat3 ࢲϯǴᆢ࡭༸ಒझ‫ޑ‬Ծ‫׳ך‬ཥૈΚ‫ک‬ӭૈ‫܄‬Ƕ Stats (signal transducer and activator of transcription) ೸ၸJak2ۚύϟᏤ٬ ‫ځ‬ႀữለූ୷೏ᕗለϯǴ‫׎‬ԋΒᆫᡏࢲϯǴ٠ᙯၮ‫ډ‬ಒझਡϣаᇨᏤ੝౦ ‫୷ޑ܄‬Ӣಔ(30, 31)Ƕӧথ٢ᜪ୏‫ނ‬ύǴStatsԿҞ߻ࣁЗςԖΎঁৎ௼ԋ঩ ೏ዴᇡрٰǴϩձࢂStat1ǵStat2ǵStat3ǵStat4ǵStat5aǵStat5bǵStat6Ǵ‫ځ‬ ϩηໆϟ‫ܭ‬75~95 KDaϐ໔ǹԶStats‫ڀ‬ԖѤঁӅӕߥ੮୔ୱǴх֖ΑSH2 domainǵLinker domainǵDNA binding domainǵCoiled-coil domainǴ೭Ѥঁ ୔ୱᆢ࡭ΑStats ‫୷ޑ‬ҁ่ᄬ‫ک‬фૈǴԿ‫ځܭ‬C ᆄ‫ޑ‬TAD(transcriptional activation domain)߾‫ڀ‬Ԗ௴୏୷Ӣᙯᒵϐфૈ(32, 33)Ƕ ӧStats‫ޑ‬Ύঁৎ௼ԋ঩ύǴStat3ࢂgp130ૻ৲໺ሀ‫ޑ‬നЬाፓ࿯‫(ޣ‬31, 34-37)ǴୖᆶΑᇨᏤ‫ط‬ಒझ࡚‫܄‬යϸᔈ(38)ǵखज༸ಒझ‫ޑ‬ᙯᒵࢲϯ‫ک‬Ծ‫ך‬ ‫׳‬ཥ(39, 40)ǵ೸ၸLIF‫ک‬IL-6٩ᒘϸᔈ٬M1 cellsϩϯ(41-43)ǵаϷᡏϣӚԄ Ӛኬ‫ޑ‬ϸᔈ(44-46)Ƕ Ӄ߻‫زࣴޑ‬ᡉҢǴJak/Statૻ৲໺ሀ೼৩൳ЯୖᆶΑ‫܌‬Ԗಒझϣ‫ޑ‬ғ‫ނ‬ ਏᔈǴӢԜ‫׳‬уँᡉрJak/Statૻ৲໺ሀ೼৩‫ޑ‬ख़ा‫܄‬Ƕ. 3.

(12) 1.1.3 n-Butylidenephthalide (BP) ࣬ᜢғ‫܄ࢲނ‬ϐࣴ‫ز‬ Angelica sinensis ࣁゾ‫ࣽ׎‬෌‫ނ‬ឦ‫ܭ‬ӭԃғ૛ҁ෌‫ނ‬Ǵࢂ‫ॺך‬΋૓߫ᆀ ‫ޑ‬྽ᘜǴ྽ᘜਥ೽ёΕᛰǴࢂനதҔ‫ޑ‬ύᛰϐ΋Ƕ྽ᘜӧ໺಍ύ૛ᛰ္ё ྽բ΋૓ံࠔǴӧᖏ‫׉‬ύёҔ‫ံܭ‬ՈࢲՈǵፓ࿶ЗภǴዎဉ೯ߡ (47)ǹӧ ऍ୯ΨᔈҔ‫ܭ‬ᖏ‫׉‬΢൳ঁ஁ࣽੱ‫( ރ‬48)ǶҞ߻Ǵςவ྽ᘜਥ೽ϩᚆள‫ډ‬Ϥ ᅿϯӝ‫ނ‬Ǻ(E)-liguistilide, (Z)-ligustilide, (Z)-n-butylidenephthalide, palmitic acid, beta-sitosterol ‫ ک‬ferulic acid (47)Ǵ‫ځ‬ύ n-Butylidenephthalide (კΒ)ࣁന Ьाಔԋԋϩ(ຬၸ 30%)Ƕ ӧа߻‫زࣴޑ‬ύǴว౜ BP Ԗᡉ๱‫לޑ‬ဍዦբҔǴόፕӧᡏϣ‫܈‬ᡏѦࣣ ς᛾ჴ BP ёаᏤठൾ‫܄‬ဍዦғߏଶᅉǴ٠Ъ೸ၸ p53-dependent ‫ک‬ -independent ‫ૻޑ‬৲೼৩຾Չࢥఠᐒ‫ڋ‬٬ဍዦಒझ঒Ϋ(49, 50)Ƕ BP Ԗ൳ᅿᛰҔ‫ޑ‬੝‫܄‬Ǵ‫ٯ‬ӵǺ೸ၸ‫ ڋ׭‬cyclo-oxygenase ၲ‫לډ‬Ոλ݈ ‫ޑ‬բҔ(51)ǹ෧጗Ј็ภ‫(ރੱޑ‬52, 53)ǶԜѦǴஒ BP ‫่ޑ‬ᄬу΢ hydroxyl moleculesǴว౜ѬёҔ‫ે୏ܭ‬ๆ‫ރ‬ฯϯ‫ݯ‬ᕍ΢(54)Ƕ೭٤ว౜ࡰрǴӧᖏ‫׉‬ ᔈҔύ BP ࢂ΋ঁкᅈ‫׆‬ఈ٠Ъ‫ڀ‬ԖወΚ‫ޑ‬ཥ‫ל‬ᕎϯӝ‫ނ‬ǶฅԶǴBP ‫ޑ‬բ Ҕჹ‫ࢲܭ‬ϯ༸ಒझϝࢂ҂‫ޕ‬Ƕ 1.2 ࣴ‫ز‬Ҟ‫ޑ‬ ҁጇፕЎ‫زࣴޑ‬Ҟ‫׆ޑ‬ఈёа‫פ‬рவύ૛ᛰύ๧‫ڗ‬рٰ‫ޑ‬΋ᅿપϯӝ ‫ނ‬ǴҔٰᆢ࡭ ES cells ‫ ک‬iPS cells ‫ޑ‬ӭૈ‫ک܄‬Ծ‫׳ך‬ཥૈΚǴ຾΋‫ڗ؁‬ж. 4.

(13) LIFǴ٠Ъගଯౢғ iPS cells ‫ޑ‬ਏ౗ǶӢԜǴҁጇፕЎ຾Չ΋‫س‬ӈ‫ޑ‬ჴᡍǴ ᛾ჴவ Angelica sinensis ਥ೽๧‫ڗ‬рٰ‫ޑ‬પϯӝ‫ ނ‬BPǴёа೸ၸࢲϯ Jak2/Stat3 ૻ৲໺ሀ೼৩Ǵٰᆢ࡭ ES cells ‫ ک‬iPS cells ‫ޑ‬ӭૈ‫ک܄‬Ծ‫׳ך‬ཥ ૈΚǴ٠Ъ᛾ჴ๏ϒ BP ёаቚуౢғ iPS cells ‫ޑ‬ਏ౗Ƕ. 5.

(14) ಃΒക ‫׷‬਑ᆶБ‫ݤ‬ 2.1 ಒझ୻Ꭶ 2.1.1 ԴႵखजᠼᆢ҆ಒझ (Mouse Embryoic Fibroblasts) Mouse Embryoic Fibroblasts (MEF)ࢂ‫ڗ‬Ծ‫ ܭ‬C57BL / 6 ࠔ‫ س‬13.5 Ϻ‫ޑ‬ज ႵǶஒ MEF ୻Ꭶ‫֖ܭ‬Ԗ 10% FBSǴ1% penicillin-Streptomycin (Invitrogen)Ǵ 1% NEAA (Invitrogen)ǴаϷ 1% L-glutamine ‫ ޑ‬DMEM (Invitrogen) ୻Ꭶన ύǴ٠‫ܫ‬࿼ӧ 37ɗǵ5% CO2 ϐ୻ᎦጃϣғߏǴ‫ٿ؂‬Ϻඤ΋ԛ୻ᎦనǶ྽ಒ झ୻ᎦԿ 7~8 ϩᅈਔǴճҔ PBS (‫؂‬Ϧϲ֖ NaCl 8gǴKCl 0.2gǴNa2HPO4 1.44gǴKH2PO4 0.24gǴа 5N NaOH ፓ᏾ pH ॶԿ 7.4) ؑࢱ୻Ꭶҝۭ೽΋ԛ ࡕǴуΕ trypsin-EDTA ‫ܫ‬࿼ 37ɗ୻ᎦጃύբҔ 5 ϩដǴа฻ໆ୻Ꭶనύ‫ک‬ trypsin ‫ޑ‬բҔǴӆஒ֖Ԗಒझ‫୻ޑ‬Ꭶ୷౽Կค๵ᚆЈᆅǶᚆЈ (1000rpmǴ 5 ϩដ) ࡕǴ౽ନ΢మనǴуΕཥᗲ‫୻ޑ‬ᎦనǴஒಒझ֡ϬѺණǴӆа 1:3~1:4 ‫ޑ‬К‫ٯ‬ஒಒझ୻ᎦԿཥ‫୻ޑ‬ᎦҝύǶ 2.1.2 iPS ‫ ک‬mES cells ‫ ܭ‬6 well ‫୻ޑ‬ᎦҝύǴ‫ ঁ؂‬well ϩձуΕ 1mL 0.2% Gelactin (Sigma)Ǵ ٠‫ܫ‬࿼‫ ܭ‬37ɗǵ5% CO2 ϐ୻Ꭶጃϣ୻Ꭶ 30 ϩដࡕǴ౽ନ 0.2% GelactinǴ ‫ ঁ؂‬well ϩձуΕ 2mL ӣྕ‫ ޑ‬DMEM ୻ᎦనǴӆஒ Feeder cells (1×106) ှএ‫ܭ‬Ԝ୻ᎦҝύǴ౽Կ 37ɗǵ5% CO2 ϐ୻Ꭶጃϣ୻Ꭶ over nightǶ౽ନ DMEM ୻ᎦనǴуΕӣྕ‫ ޑ‬stem cell ୻Ꭶన [42.5mL 1% L-glutamine ‫ޑ‬. 6.

(15) DMEM (Invitrogen) + 7.5mL Hyclon FBS (Thermo) + 0.5mL NEAA (Invitrogen) + 91l -mercaptoethanol (Invitrogen) + 5l leukemia inhibitory factor (Millipore) ] Ǵ‫ܫ‬Կ 37ɗǵ5% CO2 ϐ୻Ꭶጃϣ୻Ꭶ 1 λਔǴӆஒ stem cells ୻Ꭶ‫֖ܭ‬Ԗ feeder cells ϐ୻ᎦҝϣǴ٠‫ܫ‬࿼ӧ 37ɗǵ5 % CO2 ϐ୻Ꭶ ጃϣғߏǴ‫ٿܭ‬Ϻࡕඤಃ΋ԛ୻Ꭶనϐࡕሡ‫؂‬Ϻ‫׳‬ඤ୻ᎦనǴаዴߥಒझ ғߏ‫܌‬ሡϐᎦϩк‫ى‬Ƕ 2.2 ಒझӸࢲෳ၂ (MTT Assay) 3- (4,5-dimethyl-thiazol-2-yl) -2,5-diphenyl tetrazolium bromide (Sigma)ᙁ ᆀ MTTǴࣁ໳ՅНྋ‫ ޑ܄‬tetrazolium saltǴtetrazolium bromide ࿶җࢲಒझ ಈጕᡏύϐณࣕለѐణ䁙 (mitrchondrial succinate dehydrogenases) жᖴᗋ চౢғόྋ‫ܭ‬Н‫๋ޑ‬Յ؈ᐘ‫ ނ‬formazanǴуΕ Isopropaonal ஒϐྋှǶѝԖ ࢲಒझϣϐಈጕᡏωӸӧ‫ڀ‬Ԗࢲ‫ޑ܄‬ณࣕለѐణ䁙ǴӢԜёբࣁಒझ࿶ᛰ ‫ނ‬ೀ౛ࡕӸࢲ౗‫ࡰޑ‬኱Ƕჴᡍ‫؁‬ᡯǺᝩж୻Ꭶࡕ‫ޑ‬ಒझǴ࿶ၸಒझीኧǴ ٩‫ ঁ؂‬well 5×103 ‫ޑ‬ଆ‫ۈ‬ಒझኧҞ୻Ꭶ‫ ܭ‬96-well ‫ ޑ‬culture plate ύǴ24 λ ਔࡕ౽ନচ୻ᎦనǴуΕ֖Ԗς‫ޕ‬όӕᐚࡋ N-Butylidenephthalide (BP) ‫ޑ‬ ཥᗲ୻ᎦనǴ࿶ၸόӕਔ໔ᗺ୻ᎦࡕǴ‫ ঁ؂ܭ‬well ύϩձуΕ 10l ‫ ޑ‬MTT ྋన(0.5mg/mL)Ǵ‫ܫ‬࿼ӧ 37 ɗǵ5% CO2 ϐ୻ᎦጃϣբҔ 2~4 λਔࡕǴ౽ ନӄ೽୻ᎦనǴӆ‫ ঁ؂‬well ϩձуΕ 100l ‫ ޑ‬IsopropaonalǴа 570nm ෳ‫ۓ‬ ‫֎ځ‬ӀॶǶஒуԖᛰ‫ނ‬ྋᏊ (DMSO) ‫ޑ‬ಔձբࣁჹྣಔǴ٠аჴᡍಔϐ֎ Ӏॶঁձନаჹྣಔϐ֎ӀॶǴբკीᆉрಒझ࣬ჹӸࢲ౗Ƕ. 7.

(16) 2.3 RNA ‫ޑ‬๧‫ڗ‬Ϸ cDNA ‫ޑ‬ᇙഢ ಒझ୻ᎦԿ 7~8 ϩᅈਔǴ౽ନ୻ᎦనǴуΕ 1mL TRIzol (Invitrogen)Ǵ ࠻ྕ୻Ꭶ 5 ϩដǴճҔ‫׿ڊ‬ஒಒझӄ೽‫ڊ‬ΠǴӆஒ֖Ԗಒझ‫ ޑ‬TRIzol ౽Կ 1.5mL tube ύǴкϩஒಒझѺઇࡕǴ‫ ܭ‬tube ύуΕ 0.2mL ChloroformǴ΢Π འਗ 15 ࣾǴ࠻ྕ୻Ꭶ 2~3 ϩដǴ ᚆЈ (12000gǴ15 ϩដǴ4ɗ)ࡕǴஒ΢ మన౽Կќ΋ཥ‫ ޑ‬1.5mL tube ύǴӆуΕ 0.5mL Isopropyl-alcoholǴкϩష ӝࡕǴ࠻ྕ୻Ꭶ 10 ϩដǴᚆЈ (12000gǴ10 ϩដǴ4ɗ) ࡕǴ౽ନ΢మనǴ ӆуΕ 1mL 75% ethanol-DEPCɄH2O ؑࢱ RNAǶᚆЈ (7500gǴ5 ϩដǴ4 ɗ) ࡕǴ౽ନ΢మనǴӆճҔ੿‫ޜ‬ᐒஒ RNA ֹӄଳᔿࡕǴуΕ 0.01mL ~ 0.02mL DEPCɄH2O ஒ RNA кϩӣྋǴᓯӸ‫ܭ‬-80ɗӇጃǶஒ‫ܜ‬р‫ ޑ‬RNA ‫ ܭ‬260nm Πෳ OD ॶ (ଷ೛ OD ॶ= 1 ਔǴRNA = 40 ng/l)Ǵӆ‫؂‬ᆅӚ‫ ڗ‬2 g ‫ ޑ‬RNA ٠уΕ RNA free water ٬ᡏᑈၲ 10lǴӆӚуΕ 2l (100ng/l) oligo (dT) ‫ ک‬1l (10mM) dNTP ٠Ъ࿼‫ ܭ‬65ɗϸᔈ 5 ϩដࡕ‫ז‬ೲ࿼‫ܭ‬Ӈ΢٠ spin downǴӆӚуΕ 6.5l ‫ޑ‬ϸᔈྋన [4l 5X Bufferǵ1l 0.1 M DTTǵ1l RNase outǵ0.5l SSIII (Invitrogen)] Ъ࿼‫ ܭ‬50ɗϸᔈ 30~60 ϩដࡕǴӆ࿼‫ ܭ‬75ɗ ϸᔈ 15 ϩដࡕǴspin down ٠ᓯӸ‫ܭ‬-80ɗӇጃǶ 2.4 ջਔᆫӝ䁙ೱᙹϸᔈ (Real-time Polymerase Chain Reaction) ‫ॺך‬ճҔ real-time PCR ٰୀෳ Oct4, Sox2, c-Myc, Klf4, Jak2, Stat3, LIF, EGF, IL5, IL11, EPO ‫ ک‬OSM ೭٤୷Ӣ‫߄ޑ‬౜ໆǶჴᡍ‫؁‬ᡯǺ‫ ڗ‬4l ีញၸ. 8.

(17) ‫ ޑ‬cDNA ϩձуΕԿ 96-well ϸᔈዬύǴௗ๱ϩձуΕ 6l ϸᔈྋన [0.5l 6M Forward Primer (߄΋) + 0.5l 6M Reverse Primer (߄΋) + 5l SYBR Green PCR Master mix (Roche)] Ǵ ࿶ ֡ Ϭ ష ӝ Ϸ spin down ࡕ ‫ ܫ‬Ε StepOnePlus Real-Time PCR System (Applied Biosystems) ‫ޑ‬ሺᏔύ࿶ 95ɗ 10 ϩដǴӆ຾Չ 40 ঁൻᕉයǺ95ɗ 15 ࣾǴ60ɗ 60 ࣾǶ่‫ࡕ״‬ǴႣෳ‫ޑ‬ cycle threshold (CT)ॶ‫ޔ‬ௗճҔ EXCEL πբ߄ٰ຾Չϩ‫݋‬Ƕ 2.5 ᡵ‫܄‬ᕗለ䁙ࢉՅ (Alkaline Phosphatase Staining) ஒ iPS cells ‫ ܈‬ES cells ୻Ꭶ‫ ܭ‬6-well ‫ ޑ‬culture plate ύǴ࿶ 48 λਔࡕ౽ ନচ୻ᎦనǴуΕ֖Ԗς‫ޕ‬όӕᐚࡋ N-Butylidenephthalide (BP) ‫ޑ‬ཥᗲ୻ ᎦనǴ࿶ၸόӕਔ໔ᗺ୻ᎦࡕǴஒ୻Ꭶన౽ନǴӆуΕ PBS ੆ዎమࢱಒझ ࡕ౽ନǴख़ፄమࢱ 2 ԛǶ྽౽ନ୻Ꭶዬϣ PBS ࡕǴϩձஒ 1mL 80% ethanol уΕ୻ᎦዬϣǴ٠‫ܫ‬࿼‫ ܭ‬4ɗ‫ ۓڰ‬2~24 λਔǶௗ๱ஒ୻Ꭶዬϣ 80% ethanol ౽ନǴ٠а Distilled Water ੆ዎమࢱಒझࡕ౽ନǴӆуΕ Distilled WaterǴ‫ܭ‬ ࠻ྕ੆‫ ݰ‬2~3 ϩដࡕ౽ନǴӆа 100mM Tris-HCl buffer (pH 8.2~8.5) ‫ྕ࠻ܭ‬ ύ੆‫ ݰ‬5 ϩដࡕ౽ନࡕǴ٬Ҕ Leukocyte Alkaline Phosphatase kit (Vector) ࢉ Յऊ 20~30 ϩដǴ౽ନ Alkaline Phoshatase Substrate working solutionǴа 100mM Tris-HCl buffer (pH 8.2~8.5) ੆ዎమࢱࡕǴᢀჸ‫ྣܡ‬Ƕ྽҂ϩϯ‫ޑ‬ख ज༸ಒझ‫ڀ‬Ԗଯໆ alkaline phosphatase ϐࢲ‫཮܄‬և౜आՅϸᔈǴςϩϯ‫ޑ‬ख ज༸ಒझ߾ alkaline phosphatase ࢲ‫܄‬եǴआՅϸᔈ১ࣗԿคՅǶ. 9.

(18) 2.6 խࣝᑻӀࢉՅ (Immunofluence stain) ஒ iPS cells ‫ ܈‬ES cells ୻Ꭶ‫ ܭ‬24-well ‫ ޑ‬culture plate ύǴ࿶ 48 λਔࡕ ౽ନচ୻ᎦనǴуΕ֖Ԗς‫ޕ‬όӕᐚࡋ BP ‫ޑ‬ཥᗲ୻ᎦనǴ࿶ၸόӕਔ໔ᗺ ୻ᎦࡕǴஒ୻Ꭶన౽ନǴӆуΕ PBS ੆ዎమࢱಒझࡕ౽ନǴௗ๱уΕ 4% paraformaldehyde ‫ྕ࠻ܭ‬Π‫ ۓڰ‬10 ϩដࡕǴ౽ନ 4% paraformaldehydeǴճ Ҕ 0.1% Tween-20 / 1X PBS ੆ዎమࢱ 3 ԛǴ‫؂‬ԛ 10 ϩដǴӆуΕ 0.3% Triton-X 100 / 1X PBS ‫ྕ࠻ܭ‬ΠբҔ 30 ϩដஒಒझጢѺࢰаճࢉᏊ຾ΕǶ ӆճҔ 0.1% Tween-20 / 1X PBS ੆ዎమࢱ 3 ԛࡕǴуΕ 5% FBS / 1X PBS ‫ܭ‬ ࠻ྕΠ຾Չ blocking 2 λਔǴ຾Զᆶ 1:100 ีញࡕ‫ޑ‬΋ԛ‫ל‬ᡏ[Nanog (Novus)ǵSSEA1 (Millipore)]ӧ࠻ྕΠբҔ over nightǶճҔ 0.1% Tween-20 / 1X PBS ੆ዎమࢱ 5 ϩដࡕǴуΕ 1:500 ีញ‫ޑ‬Βԛ‫ל‬ᡏӧ࠻ྕΠᗉӀϸᔈ 1 λਔࡕǴӆа 0.1% Tween-20 / 1X PBS మࢱ 10 ϩដǴ࿶֖Ԗ DAPI ࢉᏊ ‫ ޑ‬UltraCruz™ Mounting Medium ࠾ТࡕǴջёճҔॹҥᑻӀᡉ༾᜔ᢀჸǶ 2.7 ख ज ౚ ‫ ׎ ޑ‬ԋ ‫ ک‬ϩ ϯ (Embryoid body (EB) formation and differentiation) ஒES cellsճҔ֖ԖBP‫୻ޑ‬Ꭶన୻ᎦΟжϐࡕǴҔEB formation medium [DMEM (GIBCO BRL)బу20% fetal bovine serum (FBS) (GIBCO BRL), 1mM -mercaptoethan (GIBCO BRL), 1% L-glutamin (GIBCO BRL), 1% insulin-transferin-selenium-Gsupplement (ITSG)(GIBCO BRL), 1% non-essential amino acids (NEAA) (GIBCO BRL)]୻ᎦԿUltra Low Cluster 10.

(19) Plate (Costar)Ǵ୻Ꭶऊ2~3ϺࡕǴES cellsߡёа‫׎‬ԋEBǶӆஒ EBҔ differentiation medium [DMEMబу20% fetal bovine serum (FBS)(GIBCO BRL), 1% L-glutamine (GIBCO BRL), 1% non-essential amino acids (NEAA)(GIBCO BRL), 1% penicillin and streptomycin (p/s)(GIBCO BRL), 1mM -mercaptoethanol (GIBCO BRL)]୻ᎦԿ٣Ӄςcoating Gelactin‫ޑ‬ 24-wellύǴ‫ঁ؂‬wellऊ୻Ꭶ5~6ᗭ‫ޑ‬EBǴ୻Ꭶऊ3ϺǴ٬EBёаϩϯԋΟঁ खቫ‫ޑ‬ಒझ Ƕ 2.8 ಒझྋှ‫ޑނ‬ᇙഢ ಒझ࿶ᛰ‫ނ‬ೀ౛ࡕӃ౽ନᙑ‫୻ޑ‬ᎦనǴа 1X PBS మࢱ୻Ꭶዬ 2 ԛǴஒ ዬϣූᎩనᡏ֎ନଳృǶуΕ 1ml ‫ ޑ‬PBSǴ٠ճҔ‫׿ڊ‬ஒಒझ‫ڊ‬ပǴӆஒ ಒझᝌੌనவ୻Ꭶዬύ౽Կ 1.5ml ༾ໆᚆЈᆅǶᚆЈ (1000rpmǴ10 ϩដ)Ǵ ዴჴ౽ନ΢మనࡕǴуΕ 500Pl ಒझྋှ጗ፂన (cell lysis buffer) [50mM Tris-HCl (pH 7.5)Ǵ1mM EDTAǴ 0.5M NaClǴ1mM beta-MercaptoethanolǴ 1% NP40Ǵ1% GlycerolǴ1% Protein Kinase inhibitor] բҔ 30 ϩដǴ‫ ؂‬5 ϩ ដ Vortex ΋ԛǴ٬ಒझྋှనкϩషӝǴӆа 4ɗᚆЈ(13,000rpmǴ20 ϩដ)Ƕ ᚆЈࡕǴ‫ڗ‬΢మనԿཥ‫ ޑ‬1.5ml ༾ໆᚆЈᆅǶೈқ፦ᐚࡋ‫ޑ‬ෳ‫ࢂ߾ۓ‬٬Ҕ “Bio-Rad Protein Assay”Ƕа BSA ྽኱ྗࠔǴଛᇙόӕᐚࡋ኱ྗࠔ (ఊࡋᐚ ࡋǺ0ǵ0.25ǵ0.5ǵ0.75ǵ1ǵ1.25Pg/Pl)Ǵஒีញ‫ޑ‬ኬࠔೈқ፦ྋనᆶ኱ྗ ࠔӚ‫ڗ‬р 10l ࿼‫ܭ‬ཥ‫ ޑ‬1.5ml ༾ໆᚆЈᆅύǴ٠ϩձуΕ 490l ‫ ޑ‬1 ७ Bio-Rad protein assay ࢉᏊǴషӝ֡ϬǶஒ΢ॊྋనΟख़ፄϩձуԿ 96-well 11.

(20) plate ύǴ٠а‫ ߏݢ‬595nm ෳ‫֎ځۓ‬ӀॶǴ٠ਥᏵ֎Ӏॶբр኱ྗԔጕǴа R2>0.99 ‫ޑ‬኱ྗԔጕωϒаीᆉԶෳໆрೈқ፦ᐚࡋǶਥᏵኬࠔೈқ፦ྋన ‫ޑ‬ᐚࡋόӕǴуΕಒझྋှ጗ፂనϷ Laemmli sample buffer [2% SDSǴ50mM Tris-HCl (pH 6.8)Ǵ0.2mg/mL Bromphenol BlueǴ0.1M DTTǴ10% GlycerolǴ 7.5mL H2O]Ǵനಖଛᇙԋ‫ ؂‬30l ྋనύ֖Ԗ 40g ೈқ፦‫ ޑ‬loading sampleǴ ӆаଳԄу዗ᏔǴ຾Չу዗Ǵ100ɗǴ10 ϩដǴஒኬࠔᓯӸ‫ܭ‬-80ɗӇጃύ ഢҔǶ 2.9 ౷ለΜΒ✊໊—ᆫЧ౎ㄽữᏉጤᡏႝ‫ݚ‬ȐSDS-PAGEȑ SDS-PAGE (SDS-polyacrylamide gel eletrophoresis) ຾Չ߻Ӄஒྋ‫ܭ‬ sample buffer ‫ޑ‬ኬࠔ‫ ܭ‬100ɗύу዗ 3 ϩដǶҁჴᡍ࠻‫܌‬٬Ҕ‫ޑ‬ႝ‫ݚ‬೛ഢࣁ Bio-Rad SystemǶ२Ӄ‫ ע‬glass plate sandwiches း࿼ӳǴ‫ځ‬ԛଛᇙ running gel (8%) Ϸ 5% stacking gelǶӆஒ፾ໆ‫ ޑ‬8% running gel [4mL 1.5M Tris-HCl (pH 8.8)Ǵ0.2mL 10% SDSǴ4mL 40% acrylamideǴ0.2mL 10% ammonium persulfate (APS)Ǵ0.012mL TEMEǴ10.6mL H2O ] ‫ݙ‬ΕпኲǴǶ‫ݙ‬Εၲ፾ໆଯࡋ‫ޑ‬ running gel ࡕǴӧ΢य़ᇸᇸᙟ΢΋ቫΒԛᇃᚖНǶࡑ running gel Ꮙ‫ࡕڰ‬Ǵ ౽ନ΢ቫНǴஒ comb කӧ running gel ΢БǴฅࡕ‫ݙ‬Ε፾ໆ 5% stacking gel [1.5mL 0.5M Tris-HCl (pH 6.8)Ǵ0.06mL 10% SDSǴ0.75mL 40% acrylamideǴ 0.06mL 10% APSǴ0.006mL TEMEDǴ3.6mL H2O]Ƕ྽ stacking gel Ꮙ่ࡕǴ ஒႝ‫ݚ‬೛ഢးӳǴ٠౽ѐ combǶуΕ running buffer (0.025M TrisǴ0.192 M. 12.

(21) glycineǴ3.5 mM SDS)ࡕջёஒ 30Pl ኬࠔ‫ݙ‬ΕϾࢰϣǶӧ 4ɗ຾Չႝ‫ݚ‬ǴԶ ႝᓸϩձࣁǺstacking gel ਔ‫ۓڰ‬ӧ 50 ҷ੝ǹ฻‫ډ‬ኬࠔາԿ running gel ࡕ‫ڰ‬ ‫ۓ‬ӧ 110 ҷ੝Ǵ‫ډޔ‬ኬࠔၲ࣒ዟ҃ᆄ፾྽Տ࿼Ƕ 2.10 ՋБᏀᗺ‫ݤ‬ȐWestern Blotting Analysisȑ ҁჴᡍБ‫ݤ‬Ьाࢂ٩Ᏽ“ECL Western Blotting Protocols”Ƕஒಒझྋှ‫ނ‬ ‫ڗ܌‬ள‫ޑ‬΢మన଺ೈқ፦‫ۓ‬ໆϩ‫݋‬Ƕௗ๱‫ڗ‬ኬࠔ຾Չ΢ॊ‫ ޑ‬SDS-PAGEǶ ‫ڗ‬΋஭ᆶటᙯᅄೈқ፦ጤελ፾ӝ‫ ޑ‬PVDF membrane (polyvinylidene difluoride membrane)Ǵஒ‫ ܭݰ੆ځ‬100% methanol ዎᔸǴаࢲϯ PVDF membraneǴӆஒ PVDF membrane ੆‫ܭݰ‬ᙯᅄ጗ፂన [25 mM TrisǴ192 mM glycineǴ20% (v/v) methanolǴpH 8.3] ύǴӆ‫ ڗ‬2 ஭ filter paper ‫ ک‬2 ஭ Blotting Pad ੆‫ܭݰ‬ᙯᅄ጗ፂనύǶӧॄཱུ݈΢‫ ܫ‬1 ஭ Blotting Pad ‫ ک‬filter paperǴ ӆ‫ ע‬gel ‫ܫ‬΢Ǵϐࡕӆ‫ܫ‬΢ PVDF membraneǴௗ๱ӆᇂ΢ 1 ஭ filter paper ‫ ک‬Blotting PadǴӆᇂ΢ཱུ݈҅Ǵ‫ܫ‬ΕᙯᅄኲϣǶӧᙯᅄ጗ፂనύа‫ۓڰ‬Ӽ ୻ Ȑ400mAȑ ‫ ܭ‬4ɗᙯᅄ 90 ϩដǶௗ๱٬Ҕ 1X PBS/Tween-20 (PBS-T) ጗ ፂనؑࢱ PVDF membrane ऊ 1 ϩដǴӆҔ֖ 10% ಥિѪણ(Ӽ٫ಥિѪણ) ϐ PBS-T ጗ፂన຾ՉᙟᇂϸᔈǴஒ PVDF membrane ΢‫ߚޑ‬੝౦‫่܄‬ӝՏ࿼ ᙟᇂՐǴԜ blocking ‫؁‬ᡯ‫ྕ࠻ܭ‬ϸᔈ 2 ঁλਔ‫ ܈‬4ɗ႖‫ڹ‬ϸᔈǶ ౽ନ blocking ጗ፂనǴа PBS-T ጗ፂనమࢱ PVDF membrane 3 ԛǴ‫؂‬ ԛ 10 ϩដǶϐࡕуΕа PBS-T 1:1000 ีញϐ߃ભ‫ל‬ᡏ(primary antibody). 13.

(22) [phospho-Jak2 (cell signaling) ǵJak2 (cell signaling) ǵphospho-Stat3 (cell signaling)ǵStat3 (BD)]Ǵ‫ྕ࠻ܭ‬Π٬‫֡ځ‬ϬբҔ 2 λਔ(ҭё 4ɗ႖‫ڹ‬ϸᔈ)Ƕ ߃ભ‫ל‬ᡏ่ӝࡕǴӆа PBS-T ጗ፂనమࢱ PVDF membrane 30 ϩដǶௗ๱ уΕ Horseradish Peroxidase ‫่܌‬ӝ‫ޑ‬Βભ‫ל‬ᡏ(mouse 1:10000; rabbit 1:5000)Ǵ‫ྕ࠻ܭ‬ΠբҔ 1 λਔǶᒿࡕҥջа PBS-T ጗ፂనؑࢱ PVDF membrane 1 λਔǶࢱృ‫ ޑ‬PVDF membrane നࡕуΕ ECL հӀགᔈᏊ (Amershan International, England) բҔ 3 ϩដࡕǴа೸ܴ‫׫‬ቹТᇂӧ PVDF membrane ΢Ǵ٠ᇴ‫ݰ਻و‬Ǵஒ೸ܴ‫׫‬ቹТᙟᇂӳ‫ ޑ‬PVDF membrane ѳ‫ܫ‬ ‫ ܭ‬X ӀТьպύǴ٠‫ܭ‬ས‫܊‬ϣᓸΕଯགࡋۭТȐhyperfilm-MPȑ(Amershan International, Sweden)ǴаᡉቹᏊ‫ۓک‬ቹᏊᡉႽǶۭТؑࢱࡕǴӆаஏࡋϩ ‫݋‬೬ᡏϩ‫݋‬ᡉቹӧۭТ΢‫ ޑ‬bands மࡋǴ຾ԶΑှࡑෳೈқ፦‫ޑ‬խࣝࢲ‫܄‬ ߄౜Ƕ 2.11 DNA ༾ତӈϩ‫(݋‬DNA Microarray Analysis) MEF cells ࿶җ 10g/ml ‫ ک‬40g/ml ‫ ޑ‬BP ೀ౛ 24 λਔࡕǴ๧‫ڗ‬р total RNAǴӆஒ‫ځ‬ϩձ኱‫ܫۓ‬৔ጕӕՏન (Cy3)Ƕ٩Ᏽᇙ೷ቷ୘‫ ޑ‬protocol ஒኬ ࠔᆶ Agilent Mouse G3 Whole Genome Oligo 8×60K Microarray (Agilent) ຾ ՉᚇҬǶArrays ‫ޑ‬௟ඔࢂճҔ Microarray Scanner System (Agilent)ǶኧᏵ‫ޑ‬ ϩ‫ࢂ݋‬ճҔ GeneSpring GX ೬ᡏ (Agilent)Ƕ. 14.

(23) 2.12 iPS cells ғԋਏ౗‫ޑ‬ෳ‫ۓ‬ ‫ॺך‬ճҔ‫ڗ‬Ծ Pou5f1-GFP ԴႵ(The Jackson Lab)‫ ޑ‬MEF cells ٰୀෳ iPS cells ғԋ‫ޑ‬ਏ౗ǶᙁൂٰᇥǴճҔওဎౢஒखज‫ڗ‬рǴ٠Ъஒखज‫ޑ‬ᓐǵ ϣ᠌ǵѤަ‫׀ک‬ЃѐନଳృǹಔᙃǴճҔ୊Θ୊࿗Ǵӧᙯ౽‫ ֖ډ‬Trypsin ‫ޑ‬ ၂ᆅύ੃ϯϩှǶMEF cells ୻ᎦԿ‫୼ى‬ໆਔǴ‫ॺך‬ճҔӅӕᙯࢉ pcDNA-Oct4Ǵ pcDNA-Sox2Ǵ pcDNA-c-Myc ‫ ک‬pcDNA-Klf4 ‫ ډ‬Pou5f1-GFP MEF cells ύǴ‫ٿ؂‬Ϻᙯࢉ΋ԛ(ᕴӅѤԛ)Ǵჴᡍಔӧᙯࢉϐࡕ๏ϒ 20g/ml BPǶӧಃΐϺஒԜಒझ୻Ꭶ‫ ܭ‬feeder cells ΢Ǵ٠࡭ុᢀჸ GFP ໚‫ ޑ܄‬clones ‫؂ک‬Ϻ‫׳‬ඤ୻ᎦనǶӧԜჴᡍύ BP ೀ౛ϩࣁ‫ٿ‬ಔǺ΋ಔࢂ࡭ុೀ౛ 20μg/ml ‫ ޑ‬BP (BP(T+P))ǹќ΋ಔ߾ࢂѝԖ iPS cells ୻Ꭶన(BP(T))Ƕ 2.13 ಍ीϩ‫݋‬ ࣬ᜢኧᏵϐ໔ৡ౦߾٬Ҕ one-way ANOVA ϩ‫݋‬ᔠ‫ۓ‬ǶӆճҔ Tukey HSD ٣ࡕᔠ‫ݤۓ‬ϩ‫݋‬Ǵऩ p<0.05 ߾߄Ң಍ी΢Ԗᡉ๱ৡ౦Ƕ. 15.

(24) ಃΟക ࣴ‫่݀ز‬ 3.1 ύ૛ᛰ‫ޑ‬ᑔᒧ ࣁΑ‫פ‬൨ёаҔٰғԋ iPS cells ٠Ъᆢ࡭༸ಒझϐӭૈ‫ޑ܄‬ύ૛ᛰǴ‫ך‬ ॺճҔ MEF cells ϩձ࿶җ 18 ᅿόӕᐚࡋ‫ޑ‬ύ૛ᛰೀ౛ 24 λਔࡕǴӆճҔ real-time PCR ୀෳ Oct4 ‫ ک‬Sox2 ୷Ӣ߄౜ໆǶკΟᡉҢ࿶җФክ޲ǵआन ηǵғӦ໳ǵᙝ૛ǵီЦዀǵBPǵᚊखज༸ಒझǵФजዬનǵॢ๼ऄǵआ ᝯ‫ک‬፜जዬન฻ύ૛ᛰբҔࡕǴёаᡣ Oct4 ‫ ک‬Sox2 ୷Ӣ߄౜ໆᡉ๱ቚуǴ ӧ೭٤ёаᡣ Oct4 ‫ ک‬Sox2 ୷Ӣ߄౜ቚу‫ޑ‬ύ૛ᛰ္ǴBP ࢂ୤΋‫ޑ‬પϯ ‫ނ‬Ǵ‫܌‬а‫ॺך‬ᓬӃᒧ᏷ BP ଺຾΋‫؁‬௖૸Ƕ 3.2 BP ჹ MEF cells ғߏ‫ޑ‬ቹៜ MEF cells ࿶җόӕᐚࡋ‫ ޑ‬BP ೀ౛ (5g/mlǵ10g/mlǵ20g/mlǵ 40g/mlǵ80g/mlǵ160g/mlǵ320g/mlǵ640g/ml)Ǵϩձӧ 24 λਔǵ72 λਔճҔ MTT assay ‫ޑ‬БԄᔠෳ BP ჹ MEF cells ғߏૈΚ‫ޑ‬ቹៜǶკѤᡉ Ң࿶ BP ೀ౛ࡕǴ24 λਔ‫ ک‬72 λਔࣣᡉҢ 80g/mlǵ160g/mlǵ320g/mlǵ 640g/ml ‫ޑ‬ೀ౛ᐚࡋ཮ቹៜ MEF cells Ӹࢲ౗Πफ़ǴӢԜ‫ॺך‬ᒧ᏷ӧቹៜᐚ ࡋϐΠ‫ ޑ‬5g/mlǵ10g/mlǵ20g/mlǵ40g/ml ຾ՉௗΠٰ‫ޑ‬ჴᡍǶ 3.3 BP ჹ Oct4ǵSox2ǵc-Myc ‫ ک‬Klf4 ୷Ӣ߄౜ໆ‫ޑ‬ቹៜ ࣁΑෳ၂ BP ёаҔٰғԋ iPS cells ٠Ъᆢ࡭༸ಒझ‫ޑ‬ӭૈ‫܄‬೭ঁଷ ᇥǴ‫ॺך‬ஒ MEF cells ࿶җ 5g/mlǵ10g/mlǵ20g/mlǵ40g/ml ‫ ޑ‬BP ೀ౛. 16.

(25) բҔ 24 λਔࡕǴୀෳ Oct4, Sox2, c-Myc ‫ ک‬Klf4 ୷Ӣ߄౜ໆǶკϖᡉҢ MEF cells ࿶җ 10g/ml ‫ ޑ‬BP ೀ౛ࡕǴ Oct4 ‫ ک‬Sox2 ‫୷ޑ‬Ӣ߄౜ໆԖᡉ๱‫ޑ‬ቚ уǶ‫ॺך‬Ψว౜΋ঁԖ፪‫ޑ‬౜ຝǴ྽ BP ‫ޑ‬ᐚࡋගଯ(20g/ml ‫ ک‬40g/ml) ਔǴOct4 ‫ ک‬Sox2 ‫୷ޑ‬Ӣ߄౜ໆࠅؒԖᒿ๱ᐚࡋቚуԶ΢ϲǴϸϐ۳Πफ़Ƕ ᏃᆅԜਏᔈӧ 10g/ml ೀ౛ਔၨଯǴՠ 20g/ml ‫ ک‬40g/ml ೀ౛ࡕ Oct4 ‫ک‬ Sox2 ‫୷ޑ‬Ӣ߄౜ໆΨКྋᏊჹྣಔଯǶԜѦǴc-Myc ‫ ک‬Klf4 ୷Ӣ߄౜ໆᆶ ྋᏊჹྣಔ࣬КࠅؒԖᡉ๱‫ޑ‬ቹៜǶ຾΋‫؁‬ǴճҔ ES cells ϩձೀ౛όӕᐚ ࡋ‫ ޑ‬BP բҔ 72 λਔࡕǴୀෳ Oct4, Sox2, c-Myc ‫ ک‬Klf4 ୷Ӣ߄౜ໆǶკ ϤᡉҢ ES cells ࿶җ BP բҔࡕǴёаᡣ Oct4, Sox2, c-Myc ‫ ک‬Klf4 ୷Ӣ߄ ౜ܴᡉቚуǶ 3.4 BP ჹ ES ‫ ک‬iPS cells ᆢ࡭Ծ‫׳ך‬ཥૈΚ‫ޑ‬ቹៜ ྽‫ॺך‬ᢀჸ‫ ډ‬BP ೀ౛ࡕǴ཮ቚу Oct4 ‫ ک‬Sox2 ‫୷ޑ‬Ӣ߄౜ໆǴ‫ॺך‬Ψ ၂๱ѐෳ၂࿶җ BP ೀ౛ ES ‫ ک‬iPS cells ࡕǴࢂցёаᆢ࡭‫ޑޣٿ‬Ծ‫׳ך‬ཥ ૈΚ‫ک‬ӭૈ‫܄‬Ƕӧ΋૓‫ ޑ‬ES ‫ ک‬iPS cells ୻ᎦύǴబу LIF ࢂҔٰᆢ࡭ ES ‫ ک‬iPS cells ‫ޑ‬Ծ‫׳ך‬ཥૈΚ‫ک‬ӭૈ‫܄‬Ƕр‫ܭ‬೭ঁচӢǴ‫ॺך‬೛ीΑҔ BP ඹ ж LIF ٰ୻Ꭶ ES ‫ ک‬iPS cellsǴٰ᛾ჴ೭ঁଷᇥǶAP ࢉՅࢂ٬Ҕ‫ܭ‬ୀෳ ES ‫ ک‬iPS cells ӭૈ‫ޑ܄‬΋ᅿ༸ಒझ኱૶ǶკΎ AǵB ᡉҢ ES cells ϩձ࿶ 10g/mlǵ20g/mlǵ40g/ml ‫ ޑ‬BP ೀ౛ࡕǴAP ໚‫୔ޑ܄‬ୱዴჴК഍‫܄‬ჹྣ ಔ LIF OUT ቚу೚ӭǶฅԶǴES cells ࿶ 5g/ml ‫ ޑ‬BP ೀ౛ࡕǴAP ໚‫ޑ܄‬. 17.

(26) ୔ୱࠅؒԖКჹྣಔ LIF OUT ӭǶკΎ C ᡉҢ‫ܭ‬ᡉ༾᜔ᢀჸΠǴว౜่݀ ᆶ΢ॊ่݀࣬ӕǶҗ೭٤ኧᏵᡉҢǴӧ ES cells ୻ᎦύǴBP ԖૈΚᆢ࡭ ES cells ‫ޑ‬Ծ‫׳ך‬ཥૈΚ‫ک‬ӭૈ‫܄‬Ƕ࣬ӕ‫่݀ޑ‬ǴკΖ AǵB ᡉҢ iPS cells ϩ ձ࿶ 10g/mlǵ20g/mlǵ40g/ml ‫ ޑ‬BP ೀ౛ࡕǴAP ໚‫୔ޑ܄‬ୱዴჴК഍ ‫܄‬ჹྣಔ LIF OUT ቚу೚ӭǶฅԶǴiPS cells ࿶ 5g/ml ‫ ޑ‬BP ೀ౛ࡕǴAP ໚‫୔ޑ܄‬ୱࠅؒԖКჹྣಔ LIF OUT ӭǶკΖ C ᡉҢ‫ܭ‬ᡉ༾᜔ᢀჸΠǴว ౜่݀ᆶ΢ॊ่݀࣬ӕǶҗ೭٤ኧᏵᡉҢǴӧ iPS cells ୻ᎦύǴBP ΨԖૈ Κᆢ࡭ iPS cells ‫ޑ‬Ծ‫׳ך‬ཥૈΚ‫ک‬ӭૈ‫܄‬Ƕ ԜѦǴ‫ॺך‬ΨճҔ‫ځ‬д‫ޑ‬༸ಒझ኱૶ٰ᛾ܴ೭΋ঁଷᇥǶ‫ॺך‬ӧ ES cells ୻ᎦਔǴϩձҔ 5g/mlǵ10g/mlǵ20g/mlǵ40g/ml ‫ ޑ‬BP ‫ڗ‬ж LIFǶ‫ך‬ ॺճҔխࣝᑻӀࢉՅǴᢀჸ Nanog ‫ ک‬SSEA-1 ‫ޑ‬ᑻӀ߄౜ໆǴ᛾ჴ BP ࢂց Ψёа٬ ES cells ᆢ࡭Ծ‫׳ך‬ཥૈΚ‫ک‬ӭૈ‫܄‬Ƕკΐ A ᡉҢ ES cells ϩձ࿶ 10g/mlǵ20g/mlǵ40g/ml ‫ ޑ‬BP ೀ౛ࡕǴNanog ‫߄ޑ‬౜ໆዴჴК഍‫܄‬ჹ ྣಔ LIF OUT ቚу೚ӭǴԶ ES cells ࿶ 5g/ml ‫ ޑ‬BP ೀ౛ࡕǴNanog ‫߄ޑ‬ ౜ໆࠅؒԖКჹྣಔ LIF OUT ӭǶ‫ॺך‬ճҔќ΋ঁ༸ಒझ኱૶ SSEA-1 ٰ ᢀჸࢂցԖ࣬ӕ‫่݀ޑ‬Ǵҗკΐ B ᡉҢ‫่݀ޑ‬ᆶ Nanog ߄౜ໆ‫่݀ޑ‬΋ठǶ ‫ॺך‬٬Ҕ‫ޑ‬ԴႵ iPS cells ࢂٰԾ Nanog-GFP ୷Ӣᙯ෗Ⴕ(8)Ǵ‫܌‬а‫ॺך‬ Ψᙖҗᢀჸ Nanog-GFP ‫߄ޑ‬౜ໆٰ᛾ჴǶკΜ A ᡉҢ‫่݀ޑ‬ᆶ ES cells ‫ޑ‬ Nanog ߄౜ໆ࣬಄Ǵ࿶җ BP 10g/mlǵ20g/mlǵ40g/ml ዴჴК഍‫܄‬ჹྣ. 18.

(27) ಔ LIF OUT ቚу೚ӭǶკΜ B ᡉҢᆶ ES cells ‫ ޑ‬SSEA-1 ߄౜ໆΨ࣬྽΋ ठǶ ࣁΑ຾΋‫؁‬᛾ჴǴ BP ዴჴёаᆢ࡭༸ಒझ‫ޑ‬ӭૈ‫܄‬Ǵ‫ॺך‬ճҔ 10g/ml ‫ ޑ‬BP ‫ڗ‬ж LIF ୻Ꭶ ES cells Οжϐࡕ٬ϐ‫׎‬ԋखजౚǴ຾Զϩϯ‫׎‬ԋΟख ቫಒझǴх֖ઓ࿶ಒझǵԼԺಒझǵϣखቫಒझ฻฻ǶճҔխࣝᑻӀࢉՅ ϩ‫݋‬᛾ჴ೭٤όӕᜪࠠ‫ޑ‬ಒझӧ٬ҔаΠ‫ޑ‬኱૶ࢉՅࡕԖ߄౜ǶTuj1 ȐѦ खቫ኱૶ȑ Ǵ-SMAȐύखቫ኱૶ȑ ǴGata4Ȑϣखቫ኱૶ȑ ȐკΜ΋ȑ Ƕ 3.5 BP ჹ MEF cells ϩηૻ৲໺ሀ೼৩‫ޑ‬ቹៜ җ߻य़‫ޑ‬ჴᡍ่݀ள‫ޕ‬ǴBP ёаቚу Oct4 ‫ ک‬Sox2 ୷Ӣ߄౜ໆǴΨё а٬ ES ‫ ک‬iPS cells ᆢ࡭Ծ‫׳ך‬ཥૈΚ‫ک‬ӭૈ‫܄‬ǴӢԜ‫ॺך‬၂კ຾΋‫פ؁‬р BP ࢂ೸ၸব٤ϩηૻ৲໺ሀ೼৩Ǵቹៜ ES ‫ ک‬iPS cells Ծ‫׳ך‬ཥૈΚ‫ک‬ӭ ૈ‫܄‬Ƕ ‫ॺך‬ஒ MEF cells ϩձೀ౛ 10g/ml ‫ ک‬40g/ml ‫ ޑ‬BP բҔ 24 λਔǴ຾ Չ DNA Microarray ϩ‫݋‬ǴࣁΑᙶమ BP ‫ޑ‬բҔᐒ‫ڋ‬Ǵ‫ॺך‬ճҔ KEGG pathways ၗ਑৤ϩ‫୷݋‬Ӣ߄౜‫ޑ‬ኳԄǶ߄ΒᡉҢ MEF cells ࿶ BP ೀ౛ࡕǴ ёаፓ࿯൳ঁૻ৲໺ሀ೼৩‫ࢲޑ‬ϯǴനᡉ๱‫߻ޑ‬ΟӜϩձࢂǺPPAR signaling pathwaysǵECM-receptor interaction ‫ ک‬Jak-Stat signaling pathwaysǶୖԵ೚ӭ යтள‫ ޕ‬Jak-Stat signaling pathways ᆶ stem cells നࣁ࣬ᜢǴӢԜ‫ॺך‬ᒧ᏷ Jak-Stat signaling pathways ଺຾΋‫؁‬௖૸Ƕ. 19.

(28) 3.6 BP ჹ Jak2 ‫ ک‬Stat3 ୷Ӣ߄౜ϐቹៜ ӧ༸ಒझ࣬ᜢ Jak-Stat signaling pathways ύǴJak2 ‫ ک‬Stat3 ࢂᆢ࡭༸ಒ झӭૈ‫ޑ܄‬നЬाፓ࿯ϐ୷ӢǶ‫ॺך‬ஒ ES cells ࿶җ 5g/mlǵ10g/mlǵ 20g/mlǵ40g/ml ‫ ޑ‬BP ೀ౛բҔ 72 λਔࡕǴճҔ real time PCR ୀෳ Jak2 ‫ ک‬Stat3 ୷Ӣ‫߄ޑ‬౜௃‫׎‬ǶკΜΒᡉҢ ES cells ࿶җ BP ೀ౛ࡕǴว౜ BP 10g/ml ‫ޑ‬ᐚࡋ٬ Jak2 ‫ ک‬Stat3 ϐ୷Ӣቚуਏ݀നࣁᡉ๱Ƕ 3.7 BP ჹ Jak2 ‫ ک‬Stat3 ೈқ፦߄౜ϐቹៜ ‫ॺך‬ୀෳΑ Jak2 ‫ ک‬Stat3 ୷Ӣ‫߄ޑ‬౜௃‫׎‬Ǵว౜ BP Ψ཮ቹៜ Jak2 ‫ک‬ Stat3 ୷Ӣ߄౜ໆቚуǶ຾΋‫؁‬Ǵ‫ॺך‬ճҔՋБᏀᗺ‫ݤ‬ϩ‫݋‬ǴES cells ࿶җ 5g/mlǵ10g/mlǵ20g/mlǵ40g/ml ‫ ޑ‬BP ೀ౛բҔ 72 λਔࡕǴJak2 ‫ ک‬Stat3 ೈқ፦߄౜௃‫׎‬Ƕ(კΜΟ A) ES cells ࿶җ BP ೀ౛բҔ 72 λਔǴJak2 ‫ک‬ phosphorylation-Jak2 ‫ޑ‬ೈқ፦߄౜ໆᆶ഍‫܄‬ჹྣಔ LIF OUT КၨԖܴᡉቚ уǶკΜΟ B ᡉҢ࣬ӕ‫่݀ޑ‬ǴES cells ࿶җ BP ೀ౛բҔ 72 λਔǴStat3 ‫ ک‬phosphorylation-Stat3 ೈқ፦߄౜ໆᆶ഍‫܄‬ჹྣಔ LIF OUT КၨΨԖܴᡉ ቚуǶ 3.8 BP ჹ cytokine ‫୷ޑ‬Ӣ߄౜ϐቹៜ җ΢ॊ‫ޑ‬ኧᏵᡉҢǴ༸ಒझ࿶җ BP ೀ౛ࡕǴёаࢲϯ Jak2-Stat3 signaling pathwaysǶࣁΑ൨‫ פ‬BP ࢂ೸ၸব΋ঁ cytokineǴ຾Զࢲϯ Jak2-Stat3 signaling pathwaysǴ‫ॺך‬ճҔ real time PCR ٰϩ‫݋‬ፓ௓ Jak2-Stat3 signaling. 20.

(29) pathways ‫ޑ‬΢ෞ cytokine ϐ୷Ӣ߄౜ໆǶკΜѤᡉҢஒ ES cells ࿶җ 5g/mlǵ10g/mlǵ20g/mlǵ40g/ml ‫ ޑ‬BP ೀ౛բҔ 72 λਔࡕǴୀෳ LIF, EGF, IL5, IL11, EPO ‫ ک‬OSM ୷Ӣ‫߄ޑ‬౜ໆǶว౜ ES cells ࿶җ 10g/ml ‫ޑ‬ BP ೀ౛բҔ 72 λਔࡕǴLIF, EGF, IL5, IL11, EPO ‫ ک‬OSM ୷Ӣ‫߄ޑ‬౜ໆᆶ ഍‫܄‬ჹྣಔ LIF OUT ࣬КዴჴԖᡉ๱‫ޑ‬ቚуǶ 3.9 BP ჹ iPS cells ғԋϐቹៜ җа΢‫ޑ‬ኧᏵᡉҢǴBP ёаቚу Oct4 ‫ ک‬Sox2 ୷Ӣ߄౜ໆǴԶЪёа ᆢ࡭༸ಒझ‫ޑ‬Ծ‫׳ך‬ཥૈΚ‫ک‬ӭૈ‫܄‬ǶӢԜǴ‫ॺך‬ճҔ MEF cells ᙯࢉ Oct4, Sox2, c-Myc, Klf4 Ѥঁ୷ӢǴӕਔуΕ BP ၂კቚуౢғ iPS cells ‫ޑ‬ਏ౗Ƕ ‫ॺך‬٬Ҕ‫ޑ‬ԴႵ MEF cells ࢂٰԾ Oct4-GFP ୷Ӣᙯ෗ႵǴ‫܌‬а‫ॺך‬Ψᙖҗ ᢀჸ Oct4-GFP ‫߄ޑ‬౜ໆٰ᛾ჴǶკΜϖᡉҢ MEF cells ࿶ 20g/ml BP ೀ౛ ࡕǴOct4-GFP ‫߄ޑ‬౜ໆዴჴК഍‫܄‬ჹྣಔቚу೚ӭǴԶ BP(T+P) (seeding ࡕ࡭ុу BP)ѝԖӧಃϤϺ Oct4-GFP ‫߄ޑ‬౜ໆК BP(T) (seeding ࡕό࡭ុ у BP)ᡉ๱Ǵ೭Ψཀ‫ښ‬๱Ǵӧᙯࢉ‫ޑ‬ၸำύуΕ BP ೀ౛཮ቚу iPS cells ғ ԋ‫ޑ‬ਏ౗Ƕ. 21.

(30) ಃѤക ૸ፕ ༸ಒझёа຾ՉԾ‫׳ך‬ཥ‫ک‬ϩϯԋӭᅿಒझᜪࠠǴ٠ЪᔈҔ‫ܭ‬ӭᅿᖏ‫׉‬ ‫ݯ‬ᕍ(55)Ƕ‫ૈॺך‬຾ՉᡏѦ୻Ꭶ‫܌ޑ‬Ԗ༸ಒझύǴES cells ‫ڀ‬Ԗӭૈ‫ک܄‬ϩ ϯԋΟঁЬाखቫಒझ‫ޑ‬੝‫܄‬Ǵ೭٤ख़ा‫ޑ‬੝‫܄‬ёаౢғ଼ந‫ޑ‬ಒझҔ‫ܭ‬ ᖏ‫ݯ׉‬ᕍǶӧ 2006 ԃǴВҁ‫ ޑ‬Takahashi ი໗ᙖҗᙯࢉ Oct4, Sox2, c-Myc ‫ ک‬Klf4 ೭Ѥঁ୷Ӣ຾Εᡏಒझౢғ iPS cells (8)ǴԜ iPS cells ёаլܺ ES cells ‫܌‬य़ᖏ‫ঁٿޑ‬ख़ाϐୢᚒǺխࣝ௨Ѿϸᔈ‫ک‬ᙴᕍၰቺǴӧ҂ٰѬ཮ԋ ࣁ΋໨ߚதख़ा‫ޑ‬ಒझ‫ݯ‬ᕍϐ‫ೌמ‬ǶՠࢂǴES ‫ ک‬iPS cells ‫୻ޑ‬ᎦѸ໪ᙖҗ LIF ٰࢲϯ Jak-Stat pathwayǴԶᆢ࡭‫ځ‬ӭૈ‫(܄‬39)ǶԜѦǴiPS cells ғԋਏ ౗ߚதեࢂҞ߻ iPS cells ‫ޑೌמ‬നεज़‫ڋ‬Ƕӧҁࣴ‫ز‬ύǴ‫ॺך‬ว౜΋ঁપ ϯӝ‫ނ‬ό໻ёа‫ڗ‬ж LIF ٰ୻Ꭶ ES ‫ ک‬iPS cellsǴԶЪᗋёаቚу iPS cells ғԋ‫ޑ‬ਏ౗Ƕ BP ࢂவ྽ᘜ๧‫ޑڗ‬΋ᅿપϯӝ‫ނ‬ǴѬԖᡉ๱‫לޑ‬ᕎբҔǴӧᡏϣ‫ک‬ᡏ Ѧ೿ёаᏤठൾ‫܄‬တဍዦғߏଶᅉ‫ک‬঒Ϋ(49, 50)ǶฅԶǴBP ‫ל‬ဍዦբҔ‫ޑ‬ Ԗਏᐚࡋຬၸ 50 g/ml ࣗԿၲ‫ ډ‬100 g/ml (56, 57)Ǵՠեᐚࡋϐ BP ӧ‫ݯ‬ᕍ ΢ᐒ‫ڋ‬ᆶфૈϝࢂόܴǶࣁΑෳ၂եᐚࡋϐ BP ‫ݯ‬ᕍ΢Ԗόӕ‫ޑ‬ᐒ‫ڋ‬Ǵ٠Ъ ว౜եᐚࡋϐ BP Ԗཥ‫ޑ‬фૈǴ‫ॺך‬٬Ҕե‫ ܭ‬40 g/ml ‫ ޑ‬BP ຾Չ΋‫س‬ӈ‫ޑ‬ ჴᡍǶMTT ϩ‫݋‬ว౜࿶җե‫ ܭ‬40 g/ml ‫ ޑ‬BP ೀ౛ࡕǴ٠ؒԖᏤठಒझԝ Ϋ(კѤ)ǶਥᏵ೭٤ኧᏵǴ‫ॺך‬аΠჴᡍ٬Ҕե‫ ܭ‬40 g/ml ‫ ޑ‬BP ೀ౛Ǵ೭. 22.

(31) Ψࢂ BP ٬Ҕ‫ܭ‬༸ಒझ‫ޑ‬२ԛࣴ‫ز‬Ƕ ௗΠٰ‫ॺך‬၂კ‫פ‬р΋ঁவύ૛ᛰ๧‫ޑڗ‬પϯӝ‫ނ‬Ǵ٠Ъёаቚуౢғ iPS cells ‫ޑ‬ਏ౗ǶਥᏵ೭ঁচӢǴ‫ॺך‬ճҔ 18 ᅿύ૛ᛰٰೀ౛ MEF cellsǴ ٠Ъୀෳೀ౛ࡕ MEF cells ‫ ޑ‬Oct4 ‫ ک‬Sox2 (8)ϐ୷Ӣ߄౜Ǵӧ೭ 18 ᅿύ૛ ᛰύǴBP ࢂ୤΋‫ޑ‬પϯ‫ނ‬Ǵ٠Ъё٬ Oct4 ‫ ک‬Sox2 ୷Ӣ߄౜ቚуǶკϖᡉ ҢǴ‫ॺך‬٬Ҕ MEF cells ࿶ 10 g/ml ‫ ޑ‬BP ೀ౛ࡕǴOct4 ‫ ک‬Sox2 ୷Ӣ߄౜ ࣣԖቚуǴԖ፪‫ࢂޑ‬೭ᅿբҔӧեᐚࡋ(10 g/ml)Кଯᐚࡋ(20 g/ml ‫ ک‬40 g/ml)‫ޑ‬ਏܴ݀ᡉǶа߻‫زࣴޑ‬ύǴBP ‫לޑ‬ဍዦਏᔈǴᏤठൾ‫܄‬တዦғߏ ଶᅉ‫ک‬঒Ϋ‫ޑ‬Ԗਏᐚࡋຬၸ 50 g/ml (49, 50)ǶᗨฅǴ‫زࣴॺך‬ύ٬Ҕ‫ޑ‬ᐚ ࡋե‫ ܭ‬40 g/mlǴԶЪ‫ॺך‬Ψෳ၂ၸ BP ೭٤ᐚࡋ٠ό཮೷ԋಒझԝΫǴՠ ‫ॺך‬ᇡࣁ 20 g/ml ‫ ک‬40 g/ml ‫ޑ‬ᐚࡋೀ౛Π཮Ꮴठಒझό଼நǶӢԜǴ10 g/ml ‫ ޑ‬BP ࢂᡣ Oct4 ‫ ک‬Sox2 ୷Ӣ߄౜ቚу‫ޑ‬നԖਏᐚࡋ(კϖ)ǶBP ёа ቚу Oct4 ‫ ک‬Sox2 ୷Ӣ߄౜ǴӢԜ‫ॺך‬ᗋෳ၂ճҔ BP ٰቚу iPS cells ‫ޑ‬ਏ ౗ǶӵკΜϖᡉҢǴ‫ॺך‬ዴ‫ ۓ‬BP ೀ౛ࡕёаቚу iPS cells ғԋ‫ޑ‬ਏ౗ǴԶ Ъჹྣಔᆶ௓‫ڋ‬ಔ࣬КऊගଯΑϖ७‫ޑ‬ਏ౗Ƕӧ҂ٰ‫زࣴޑ‬ύǴ‫ॺך‬ஒ‫פ‬ р‫׳‬ӭ‫ڀ‬Ӽӄ‫ک܄‬ගଯ iPS cells ғԋਏ౗‫ޑ‬Б‫ݤ‬Ƕ ӧа۳‫زࣴޑ‬ǴOct4 ‫ ک‬Sox2 ёаᆢ࡭ ES cells ‫ޑ‬ӭૈ‫(܄‬58)ǶӢԜǴ ‫ڀޑزࣴॺך‬ᡏҞ኱ࣁǺ1.ෳ၂ BP ёаᆢ࡭ ES ‫ ک‬iPS cells ‫ޑ‬Ծ‫׳ך‬ཥૈ Κ‫ک‬ӭૈ‫܄‬ǹ2.ዴ‫ ۓ‬BP ᆢ࡭༸ಒझӭૈ‫ޑ܄‬ᐒ‫ڋ‬Ƕ‫ॺך‬ճҔ BP ‫ڗ‬ж LIF. 23.

(32) ٰ୻Ꭶ ES celltǴว౜ BP Ψёаᡣ Oct4, Sox2, c-Myc ‫ ک‬Klf4 ୷Ӣ߄౜ܴ ᡉቚу(კϤ)Ƕ٠Ъ٬Ҕ BP ٰ୻Ꭶ ES ‫ ک‬iPS cellsǴ‫ॺך‬ҔΑ൳ঁ༸ಒझ኱ ૶Ǵх֖ AP ࢉՅǵNanog ‫ ک‬SSEA1Ǵٰዴ‫ ۓ‬BP ёа‫ڗ‬ж LIF ٰ୻Ꭶ ES ‫ ک‬iPS cellsǴЀ‫ ࢂځ‬10 ‫ ک‬20 g/ml (კΎǵΖǵΐǵΜ)Ǵ೭ঁኧᏵᆶ Oct4 ‫ ک‬Sox2 ୷Ӣ߄౜‫ޑ‬ኧᏵ΋ठǶԜѦǴ‫ॺך‬ճҔ 10 g/ml ‫ ޑ‬BP ‫ڗ‬ж LIF ٰ ୻Ꭶ ES ‫ ک‬iPS cellsǴ࿶ၸΟж୻ᎦࡕǴճҔ embryoid body (EB)‫׎‬ԋ೭ঁ ‫(ೌמ‬3, 8)Ǵٰෳ၂༸ಒझ‫ޑ‬ӭૈ‫܄‬Ǵ೭ঁኧᏵࡰрǴBP ёа‫ڗ‬ж LIF ٰ୻ Ꭶ ES ‫ ک‬iPS cellsǶ ࣁΑࣴ‫ ز‬BP ೀ౛ࡕ‫ځ‬բҔ‫ޑ‬ᐒ‫ڋ‬Ǵ‫ॺך‬ճҔ microarray ϩ‫פ݋‬р BP Ьाፓ࿯‫ޑ‬೼৩Ƕ߻ΟӜ‫ޑ‬೼৩ǺPPAR signaling pathwayǵ ECM-receptor interaction ‫ ک‬Jak-Stat signaling pathway (߄Β)Ƕӧ 2010 ԃǴMo et al.ࣴ‫ز‬ᡉ Ң PPAR ፓ࿯ LIF ೸ၸ Tyk2-Stat3 ೼৩ǴᇨᏤԴႵ‫ޑ‬खज༸ಒझғߏᆶԾ ‫׳ך‬ཥ(59)Ƕ‫ॺך‬ୀෳΑ ES cells ࿶ BP ೀ౛ࡕ PPAR ‫୷ޑ‬Ӣ߄౜Ǵว౜ PPAR ୷Ӣ߄౜ᆶ௓‫ڋ‬ಔ࣬К٠ؒԖৡ౦ǴਥᏵ೭ঁচӢǴ‫ॺך‬ஒ๱ख़‫ܭ‬ Jak-Stat signaling pathway Ƕ ‫ ॺ ך‬ዴ ᇡ ༸ ಒ झ ࿶ BP ೀ ౛ ࡕ Ǵ phosphorylation-Jak2 ‫ ک‬phosphorylation-Stat3 ‫ޑ‬ೈқ፦߄౜ቚу(კΜΟ)Ƕ LIF ‫ૻޑ‬৲೸ၸ Jak ࢲϯΑ࣬ᜢ‫ૻޑ‬৲໺ሀǴ٠Ъࢲϯ Stat3 (39)Ǵჹ‫ ܭ‬ES cells ‫ޑ‬Ծ‫׳ך‬ཥૈΚ LIF ࢂ‫ת‬ᄽ΋ঁόё‫܈‬લ‫فޑ‬Յ(31)Ƕ‫ޑॺך‬ኧᏵࡰ рǴ࿶ BP ೀ౛ёаࢲϯ Jak2 ‫ ک‬Stat3 ٬ ES cells ᆢ࡭Ծ‫׳ך‬ཥૈΚǶԜѦǴ. 24.

(33) Jak-Stat signaling pathway ёа೸ၸ LIF, EGF, EPO, IL5, IL11 ‫ ک‬OSM (17-21) ೭٤ಒझᐟનࢲϯǶ‫ॺך‬ว౜ӧ࿶ၸ 10 g/ml ‫ ޑ‬BP ೀ౛ࡕǴ೭٤ಒझᐟ ન‫୷ޑ‬Ӣ߄౜೿Ԗቚу‫ޑ‬౜ຝǴ೭ঁኧᏵᆶೈқ፦߄౜‫ޑ‬ኧᏵ΋ठǴ࿶ 10 g/ml ‫ ޑ‬BP ೀ౛ࡕ߄౜ໆࢂനଯ‫ޑ‬Ƕ‫ॺך‬ዴ‫ ۓ‬BP ೸ၸࢲϯ Jak-Stat signaling pathway ࣬ᜢ‫ޑ‬ಒझᐟનǴ຾Զࢲϯ Jak2 ‫ ک‬Stat3 ‫ޑ‬ೈқ፦Ǵٰၲ‫ډ‬ᆢ࡭༸ ಒझӭૈ‫܄‬ϐфૈǶ നࡕǴ‫ॺך‬ճҔ፦ᡏᙯࢉѤঁ୷ӢǴ٠Ъ࿶җ BP ೀ౛ٰቚу iPS cells ғԋ‫ޑ‬ਏ౗Ǵਏ౗եࢂౢғ iPS cells ‫ೌמ‬ύനε‫ޑ‬ምᛖ(60)ǶӧӃ߻‫زࣴޑ‬ ύǴԖ΋ঁλϩη[valproic acid (VPA)]ёаҔٰගଯ iPS cells ‫ޑ‬ғԋਏ౗ (10)ǶVPA ٠ό཮ቚу Oct4 ‫ ک‬Sox2 ‫୷ޑ‬Ӣ߄౜ǴՠࢂѬ཮ቚуੰࢥ൪Ε ୷Ӣ‫ޑ‬ਏ౗(10)Ƕӧ 2010 ԃǴEsteban et al.‫زࣴޑ‬ᡉҢᆢғન C ёаቚуԴ Ⴕ‫ک‬Γᜪ‫ ޑ‬iPS cells ғԋਏ౗(61)Ƕ೭٤ࣴ‫ز‬ᡉҢ΋ঁλ‫ޑ‬ϯӝ‫ނ‬ёаҔٰ ቚуғԋ iPS cells ‫ޑ‬ਏ౗ǶࣁΑෳ၂೭ঁଷᇥǴ‫ॺך‬٬Ҕ pou5f1-GFP ୷Ӣ ᙯ෗Ⴕ‫ ޑ‬MEF cellsǴ٠Ъஒ Oct4, Sox2, c-Myc ‫ ک‬Klf4 ୷Ӣ໺Ε MEF cellsǴ ӆу΢ BP ೀ౛Ǵ٠ᆶ௓‫ڋ‬ಔόу BP ೀ౛‫ޑ‬Кၨ iPS cells ғԋਏ౗Ƕ‫ॺך‬ ว౜࿶ BP ೀ౛ёаቚу iPS cells ғԋǶ೭٤ኧᏵࢂ BP ёаቚу iPS cells ғԋਏ౗‫ޑ‬Ԗճ᛾ᏵǶ ‫ॺך‬ዴ‫ۓ‬Α BP ೸ၸፓ࿯ୖᆶ‫ ܭ‬Jak2-Stat3 signaling pathways ύ‫ޑ‬ಒझ ᐟનǴቚу Jak2 ‫ ک‬Stat3 ‫ޑ‬ೈқ፦߄౜Ǵᡣ༸ಒझᆢ࡭ӭૈ‫ک܄‬Ծ‫׳ך‬ཥ. 25.

(34) ૈΚǹԜѦǴBP ‫ޑ‬ೀ౛ࡕёаቚу iPS cells ғԋ‫ޑ‬ਏ౗Ƕஒٰ BP ёаว ৖ԋࣁ଼ந१ࠔǴҔٰࢲϯ༸ಒझǴԶЪӧ҂ٰ BP ёаҔ‫ׯܭ‬๓ iPS cell ғԋϐਏ౗Ƕ. 26.

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(40) კ߄. ߄΋ǵջਔ‫ۓ‬ໆᆫӝ䁙ೱᙹϸᔈ‫ׇ‬ӈ΋ំ߄ Primer. Forward SequenceǺ(5’-3’). Reverse SequenceǺ(5’-3’). Sox2. AGGGCTGGACTGCGAACTG. TTTGCACCCCTCCCAATTC. c-Myc. CATTCAAGCAGACGAGCA. CGAGTTAGGTCAGTTTATGCAC. Klf4. CCTTTCAGTGCCAGAAGT. ACTACGTGGGATTTAAAAGTGC. Jak2. CAATGATAAACAAGGGCAAATGAT. CTTGGCAATCTTCCGTTGCT. Stat3. CCCCGTACCTGAAGACCAAGT. CCGTTATTTCCAAACTGCATCA. EGF. GAGTCTGCCTGCGGATGGT. GCTGCAGGGAGGGAGACA. EPO. CCCCCACGCCTCATCTG. TGCCTCCTTGGCCTCTAAGA. IL5. TCCCTGCTACTCTCCCCAAA. CAACCTTCTCTCTCCCCAAGAA. IL11. CATGCCACACCCCAAACAA. CCCCTCACCCAGGTCTACTG. LIF. CCTACCTGCGTCTTACTCCATCA. TGTTTTCCCCAAAGGCTCAA. OSM. CGGTCCACTACAACACCAGATG. GCGATGGTATCCCCAGAGAA. ͊-Actin. GTGCGTGACATCAAAGAGAAGC. TGGATGCCACAGGATTCCATAC. Taqman-Oct4. GAGGCTACAGGGACACCTTTC. GTGCCAAAGTGGGGACCT. Roche Universal Probes 6. Taqman-͊-Actin. CTAAGGCCAACCGTGAAAAG. ACCAGAGGCATACAGGGACA. Roche Universal Probes 64. 32. Taqman Probe.

(41) ߄ΒǵճҔ KEGG ‫ ک‬Babelomics ၗ਑৤ϩ‫݋‬ᡉ๱߄౜‫୷ޑ‬ӢኧᏵᆶғ‫ނ‬ф ૈϩᜪǶ. I: number of upregulated genes; D: number of downregulated genes.. 33.

(42) კ΋ǵLIF ೸ၸࢲϯ Jak/Stat3 signaling pathwaysǴፓ௓༸ಒझ‫ޑ‬ғ‫܄ࢲނ‬Ǵ კᘏ‫ڗ‬ԾЎക(62)Ƕ. 34.

(43) კΒǵn-Butylidenephthalide ‫ޑ‬ϯᏢ่ᄬ. 35.

(44) კΟǵճҔ real-time PCR ୀෳ MEF cells ϩձೀ౛ 18 ᅿόӕᐚࡋ‫ޑ‬ύ૛ᛰ բҔ 24 λਔࡕǴOct4 ‫ ک‬Sox2 ୷Ӣ߄౜ໆǶ. 36.

(45) კѤǵճҔ MTT assay ϩ‫ ݋‬BP ჹ MEF cells ғߏૈΚ‫ޑ‬ቹៜǶMEF cells ೀ౛όӕᐚࡋ‫ ޑ‬BP ϩձբҔ 24 ‫ ک‬72 λਔǶӚಔ࣬ჹ֎Ӏॶ‫ޑ‬ѳ֡ॶᆶ኱ ྗৡǶ*P 0.05 versus control (DMSO*Ƕ. 37.

(46) კϖǵճҔ real-time PCR ୀෳ MEF cells ೀ౛όӕᐚࡋ BP բҔ 24 λਔࡕǴ Oct4, Sox2, c-Myc, Klf4 ୷Ӣ߄౜ໆǶ*P 0.05 versus control (DMSO*Ƕ!. 38.

(47) კϤǵճҔ real-time PCR ୀෳ ES cells ೀ౛όӕᐚࡋ BP բҔ 72 λਔࡕǴ Oct4, Sox2, c-Myc, Klf4 ୷Ӣ߄౜ໆǶ*P 0.05 versus control (LIF OUT).. 39.

(48) (A). (B) LIF OUT. BP 10. LIF. BP 20. BP 5. BP 40. (C). კΎǵ(A) ES cells ೀ౛όӕᐚࡋ BP բҔ 72 λਔࡕǴճҔ alkaline phosphatase staining ϩ‫݋‬९ᕒკǶ(B) Οԛᐱҥჴᡍ‫ޑ‬ѳ֡ॶr SD Ƕ*P 0.05 versus LIF OUT. (C) ES cells ೀ౛όӕᐚࡋ‫ޑ‬BP բҔ72 λਔࡕǴalkaline phosphatase staining ‫ ܭ‬40 ७ᡉ༾᜔კǶ. 40.

(49) (A). (B). (C). კΖǵ(A) iPS cells ೀ౛όӕᐚࡋ BP բҔ 72 λਔࡕǴճҔ alkaline phosphatase staining ϩ‫݋‬९ᕒკǶ(B) Οԛᐱҥჴᡍ‫ޑ‬ѳ֡ॶr SDǶ*P 0.05 versus LIF OUT. (C) iPS cells ೀ౛όӕᐚࡋ‫ ޑ‬BP բҔ 72 λਔࡕǴalkaline phosphatase staining ‫ ܭ‬40 ७ᡉ༾᜔კǶ. 41.

(50) (A) LIF. LIF OUT. 100ȫm. 100ȫm. 100ȫm. DAPI. Nanog. Merge. 100ȫm. BP 5. DAPI. 100ȫm. Nanog. 100ȫm. Merge. 100ȫm. DAPI. 100ȫm. Nanog. 100ȫm. BP 20. BP 10. Merge. 100ȫm. DAPI. 100ȫm. Nanog. 100ȫm. Merge. 100ȫm. BP 40. DAPI. 100ȫm. Nanog. 100ȫm. Merge. 100ȫm. DAPI. 100ȫm. Nanog. 100ȫm. Merge. (B) LIF. LIF OUT. 100ȫm. 100ȫm. 100ȫm. DAPI. SSEA1. Merge. 100ȫm. 100ȫm. 100ȫm. BP 5. DAPI. SSEA1. Merge. 100ȫm. 100ȫm. 100ȫm. BP 20. BP 10. DAPI. SSEA1. Merge. 100ȫm. 100ȫm. 100ȫm. DAPI. SSEA1. Merge. 100ȫm. 100ȫm. 100ȫm. BP 40. DAPI. SSEA1. Merge. 100ȫm. 100ȫm. 100ȫm. DAPI. SSEA1. Merge. კΐǵ(A) ES cells ೀ౛όӕᐚࡋ BP բҔ 72 λਔࡕǴӧᑻӀᡉ༾᜔Πᢀჸ Nanog-GFP ‫߄ޑ‬౜ໆǶ(B) ES cells ೀ౛όӕᐚࡋ BP բҔ 72 λਔࡕǴӧᑻ Ӏᡉ༾᜔Πᢀჸ SSEA1 ‫߄ޑ‬౜ໆǶ. 42.

(51) (A) LIF out. LIF. 100ȫm. BP5. 100ȫm. 100ȫm. BP10. BP20. 100ȫm. BP40. 100ȫm. 100ȫm. (B) LIF. LIF OUT. 100ȫm. DAPI. 100ȫm. 100ȫm. BP 5. DAPI. 100ȫm. SSEA1. 100ȫm. DAPI. 100ȫm. 100ȫm. Merge. 100ȫm. SSEA1. 100ȫm. DAPI. 100ȫm. SSEA1. 100ȫm. Merge. BP 20. BP 10. 100ȫm. SSEA1. 100ȫm. Merge. 100ȫm. BP 40. DAPI. DAPI. 100ȫm. SSEA1. 100ȫm. Merge. 100ȫm. SSEA1. 100ȫm. Merge. Merge. კΜǵ(A) iPS cells ೀ౛όӕᐚࡋ BP բҔ 72 λਔࡕǴӧᑻӀᡉ༾᜔Πᢀჸ Nanog-GFP ‫߄ޑ‬౜ໆǶ(B) iPS cells ೀ౛όӕᐚࡋ BP բҔ 72 λਔࡕǴӧᑻ Ӏᡉ༾᜔Πᢀჸ SSEA1 ‫߄ޑ‬౜ໆǶ. 43.

(52) Ectoderm. 50ȫm. 50ȫm. 50ȫm. Mesoderm. DAPI. Tuj-1. Merge. Endoderm. DAPI. 50ȫm. 50ȫm. 50ȫm. 50ȫm. Ƞ-SMA. 50ȫm. Merge. 50ȫm. DAPI. Geta-4. Merge. კΜ΋ǵճҔ BP ‫ڗ‬ж LIF ୻Ꭶ ES cells Οжϐࡕ٬ϐ‫׎‬ԋ EB ϩϯғԋΟ खቫಒझǶ. 44.

(53) კΜΒǵճҔ real-time PCR ୀෳ ES cells ೀ౛όӕᐚࡋ BP բҔ 72 λਔࡕǴ Jak2 ‫ ک‬Stat3 ୷Ӣ߄౜ໆǶ*P 0.05 versus LIF OUT.. 45.

(54) (A). (B). კΜΟǵ(A) ES cells ೀ౛όӕᐚࡋ BP բҔ 72 λਔࡕǴճҔ western blot ϩ‫ ݋‬Jak2 ‫ ک‬phosphorylation-Jak2 ೈқ፦߄౜ໆǶ(B) ES cells ೀ౛όӕᐚࡋ BP բҔ 72 λਔࡕǴճҔ western blot ϩ‫ ݋‬Stat3 ‫ ک‬phosphorylation-Stat3 ೈ қ፦߄౜ໆǶ. 46.

(55) კΜѤǵճҔ real-time PCR ୀෳ ES cells ೀ౛όӕᐚࡋ BP բҔ 72 λਔࡕǴ LIF, EGF, IL5, IL11, EPO, OSM ୷Ӣ߄౜ໆǶ*P 0.05 versus LIF OUT.. 47.

(56) 20 18. GFPpositiveclones. 16 14 12. 6days. 10. 8days. 8. 12days. 6. 15days. 4 2 0 control. BP(T). BP(T+P). კΜϖǵMEF cells ᙯ෗ Oct4, Sox2, c-Myc, Klf4 ӕਔуΕ BPǴϩձӧಃϤ ϺǵಃΖϺǵಃΜΒϺǵಃΜϖϺճҔᑻӀᡉ༾᜔ᢀჸ٠ीኧ Oct4-GFP positive clones ಍ीբკǶControlǺᙯ෗ Oct4, Sox2, c-Myc, Klf4ǹBP (T)Ǻ ᙯࢉ Oct4, Sox2, c-Myc, Klf4 ӧᙯࢉၸำύу BPǴϐࡕ୻Ꭶ߾όуǹBP (T+P)Ǻᙯࢉ Oct4, Sox2, c-Myc, Klf4 ӧᙯࢉၸำ‫ک‬ϐࡕ୻Ꭶࣣ࡭ុу BPǶ. 48.

(57)