博碩士論文 100324053 詳細資訊




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姓名 鄭宇鎧(Yu-Kai Cheng)  查詢紙本館藏   畢業系所 化學工程與材料工程學系
論文名稱 人類體細胞從組成誘導型多能性幹細胞培養在無飼養層上
(Reprogramming of human somatic cells into induced pluripotent stem cells under feeder-free conditions)
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摘要(中) 人類胚胎幹細胞跟人類誘導型多能幹細胞因為他們能分化成各種體細胞的特殊能力,這個能力有許多潛能可以應用在治療許多疾病上,,然而人類胚胎幹細胞和人類誘導型多能幹細胞於臨床醫學上會受限於使用老鼠成纖維母細胞當作飼養層,在此篇我們成功培養出人類誘導型多能幹細胞且沒使用老鼠成纖維母細胞當作飼養層,我們用逆轉錄病毒含有四個多能性基因轉錄人類脂肪幹細胞製造出人類誘導型多能幹細胞並培養在合成的盤子(VN-dishes),在轉錄的第四天,轉錄四個多能性基因的人類脂肪幹細胞轉移到老鼠成纖維母細胞為飼養層的盤子上當作對照組另一組則移到合成的盤子(VN-dishes)培養,在轉錄的14天之後可以清楚看到人類誘導型多能幹細胞菌落在老鼠成纖維母細胞,而人類誘導型多能幹細胞菌落在合成的盤子(VN-dishes)也能檢測到, 當 105 個人類脂肪幹細胞種在盤子上,人類誘導型多能幹細胞菌落在老鼠成纖維母細胞上大約12028個,而在合成的盤子上(VN-dishes)大約258個,人類誘導型多能幹細胞培養在合成盤子(VN-dishes)的生成效率比培養在老鼠成纖維母細胞上還要低,不過在合成盤子(VN-dishes)上的人類誘導型多能幹細胞菌落可以顯示鹼性磷酸酶(AP staining),用免疫螢光染色也可以清楚的表現出SSEA-4的多能性蛋白,這些都能指出我們成功地製造出人類誘導型多能幹細胞培養在無飼養層的合成盤子(VN-dishes)上。
摘要(英) Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) have potentially therapeutic applications in the treatment of many diseases, due to their unique ability to differentiate into any type of somatic cell. However, the clinical potential of hESCs and hiPSCs is restricted by the use of mouse embryonic fibroblasts (MEFs) as a feeder layer for these cells. I found that hiPSCs can be successfully generated without the use of a feeder layer of MEFs. hiPSCs were generated by transducing human adipose-derived stem cells (hADSCs) with a retrovirus containing pluripotency genes, and the hiPSCs were cultured on synthetic dishes grafted with an oligopeptide derived from vitronectin (VN-dish). On the fourth day after transduction, the hADSCs transduced with pluripotency genes were transferred to a MEF layer for culturing as a control condition or to VN-dishes for culture. The hiPSC colonies in the MEF-cultures were clearly observed at day 14 after transduction, whereas hiPSC colonies were detected on the VN-dishes after the cells were passaged. When 105 hADSCs were seeded on the dishes, the number of colonies generated on the MEFs was 12028, while the number of colonies generated on VN-dishes was 258. Thus, the efficiency of hiPSC generation on the VN-dishes under feeder-free conditions was lower than hiPSCs cultured on MEFs. However, the hiPSC colonies from VN-dishes demonstrated alkaline phosphatase activity, and immunohistochemistry suggested that the hiPSCs generated on VN-dishes expressed the pluripotency protein, stage-specific embryonic antigen-4 (SSEA-4), under feeder-free conditions.
關鍵字(中) ★ 誘導型多能性幹細胞 關鍵字(英) ★ induced pluripotent stem cells
論文目次 Index of content
Chapter 1: Introduction 1
1-1 Stem Cells 1
1-1-1 Self Renewal and Lineage Plasticity 2
1-1-2 Totipotency and Nuclear Transfer 3
1-1-3 Pluripotent Stem Cells and Induced Pluripotent Stem Cells 6
1-1-4 Adult Stem Cells 7
1-2 Reprogramming Method 8
1-3 Characterization of Pluripotent Stem Cells 10
1-3-1 Colony formation 10
1-3-2 Alkali phosphatase activity 10
1-3-3 Pluripotent gene Expression 12
1-3-4 Pluripotent protein Expression 12
1-3-5 Differentiation ability 12
Chapter 2: Materials and Methods 14
2-1 Material 14
2-1-1 Cell line 14
2-1-2 Chemical 14
2-1-3 Medium 15
2-2 Cell Isolation 15
2-2-1 Human adipose-derive stem cell (hADSCs) 15
2-2-2 Mouse embryonic fibroblasts (MEFs) 16
2-3 Cell Culture 19
2-3-1 Culture of hADSCs and MEFs 19
2-3-2 Culture of 293FT cell line 19
2-3-3 Culture of human pluripotent stem cell lines 20

2-4 Amplification plasmid…………………………...…………………………….24
2-4-1 Bacteria transformation 23
2-4-2 Plasmid Amplification 23
2-4-3 Plasmid purification 23
2-5 Transfection of 293FT cells 24
2-6 The generation of induced pluripotent stem cells 25
2-6-1 Culture hiPS on MEFs 25
2-6-2 Culture hiPS on VN-dish 26
2-7 Immunofluorescence 26
2-8 Colony attachment ratio 28
2-9 Alkaline phosphatase live staining assay 28
Chapter 3: Results and Discussion 30
3 Reprogramming of hADSCs into hiPSCs 30
3-1 Transfection of 293FT cells and generation of retrovirus containing pluripotent gene 30
3-2 Transduction of hADSCs into hiPSCs 31
3-3 Reprogramming of hADSCs into hiPSCs 32
3-4 Characterization of hiPSCs 34
Chapter 4: Conclusion 36
References 37
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指導教授 樋口亞紺(Akon Higuchi) 審核日期 2015-7-14
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