小鼠胚胎幹細胞株之建立及人類誘導多能性幹細胞之培養技術

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姓名

王婷(Ting Wang) 查詢紙本館藏

畢業系所

系統生物與生物資訊研究所

論文名稱

小鼠胚胎幹細胞株之建立及人類誘導多能性幹細胞之培養技術
(Establishment of mouse embryonic stem cell lines and Technology of human induced Pluripotent Stem cells culture)

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摘要(中)

胚胎幹細胞是來自哺乳動物胚胎期之囊胚的內細胞團，擁有長時間不會分化的能力，同時保持多能性，並且能夠分化成三個胚層的各種細胞。在一定條件下，多能性幹細胞可以在體外無限地繁殖並保持分化成各種體細胞和胚胎外組織的能力。而從人類或動物的成體細胞中轉殖基因Oct3 / 4，SOX2 ，c - Myc，Klf4，Nanog和LIN28於細胞內後，在培養胚胎幹細胞的條件下，也可培養成為誘導多能性幹細胞(iPS cells)，這些細胞展現出似胚胎幹細胞的型態與生長特徵及胚胎幹細胞的標的基因。在我們的研究中，我們利用小鼠受精後的受精卵，成功地在體外培養小鼠胚胎幹細胞。利用免疫螢光染色確定多能性因子Oct3 / 4、SOX2存在於我們所建立的小鼠胚胎幹細胞中。我們使用人類胎兒肺部纖維母細胞、月經血細胞及子宮內膜細胞，以基因殖入的方法將其倒程式化(reprogramming) 後誘導成多能性幹細胞。最後這些細胞也經由免疫螢光染色確定多能性因子Oct3 / 4、SOX2及人類幹細胞專一性抗體SSEA4、TRA-1-60的存在。我們成功地建立了小鼠胚胎幹細胞及人類誘導多能性幹細胞技術，並希望利用這些技術在幹細胞生物、腫瘤醫學及組織工程醫學的研究領域發揮重要貢獻和作用。

摘要(英)

Embryonic stem (ES) cells are derived from the inner cell mass of mammalian blastocysts. These cells have not only the ability to maintain pluripotency, but also the ability
to differentiate into cells of all three germ layers. Under certain conditions, pluripotential stem cells can be stimulated to propagate indefinitely in vitro and still maintain the capacity for differentiation into a wide variety of somatic and extra embryonic tissues. Recently, induction of pluripotent stem cells have been derived under ES cell culture conditions human or mouse embryonic ﬁbroblasts and adult ﬁbroblasts by introducing factors such as Oct3/4, Sox2, c-Myc, Klf4, Nanog and LIN28 into the culture medium. These cells, which are defined as
induced pluripotent stem (iPS) cells, exhibit the morphological and growth properties of ES cells and express ES cell marker genes. In this study, we established the ES cell techniques to derive mouse ES from fertilized eggs in vitro. We also develop the method of inducing and culturing the "human lung fetal fibroblast, endometrium cell and uterine endometrium cells" to become human iPS cells with the method of infecting four genes into these cells to allow cell reprogramming to pluripotency. To identify these hiPS cells, we performed immunocytochmistry. The pluripotent factors such as Oct3/4, SOX2 and Nanog, and human specific markers TRA-1-60 and SSEA-4, were markedly expressed in each human iPS cells. We prospect that these stem cell techniques will be invaluable for future researches of stem cell biology, cancer medicine and tissue engineering.

關鍵字(中)

★ 人類誘導多能性幹細胞
★ 胚胎幹細胞株

關鍵字(英)

★ induced Pluripotent Stem cells
★ embryonic stem cell

論文目次

摘要 .......................................................................................................................... i
Abstract ................................................................................................................................ ii
致謝 ...................................................................................................................... iii
圖目錄 .......................................................................................................................... vi
中英名詞對照表 ...................................................................................................... vii
第一章緒論 ......................................................................................................................... 1
1-1 前言 ................................................................................................................. 1
1-2 胚胎幹細胞與成體幹細胞 ............................................................................... 3
1-2-1 胚胎幹細胞 ............................................................................................ 3
1-2-2 成體幹細胞 ............................................................................................ 5
1-2-3 成體幹細胞與胚胎幹細胞現今發展與未來應用 ................................... 5
1-3 誘導多能性幹細胞 .......................................................................................... 6
1-3-1 誘導多能性幹細胞意義與技術.............................................................. 6
1-3-2 誘導多能性細胞的倒程式化 ................................................................. 7
1-3-3 誘導多能性幹細胞現今發展與未來應用 .............................................. 7
第二章實驗材料與方法 ...................................................................................................... 9
2-1 小鼠胚胎幹細胞株之建立 ............................................................................... 9
2-1-1 餵養細胞培養及處理 ............................................................................. 9
2-1-2 小鼠胚胎幹細胞之培養與維持............................................................ 10
2-2 人類誘導多能性幹細胞培養與維持 ............................................................. 13
2-2-1 反轉錄病毒製成與轉染 ....................................................................... 14
2-2-2 人類誘導多能性幹細胞培養及繼代方法 ............................................ 15
2-3 幹細胞免疫螢光染色..................................................................................... 17
第三章結果 ....................................................................................................................... 18
3-1 小鼠胚胎幹細胞型態及成果 ......................................................................... 18
3-2 人類誘導多能性幹細胞型態及成果 ............................................................. 20
3-2-1 MRC-5(human lung fetal fibroblast)iPS 細胞 ....................................... 20
3-2-2 Edom(human endometrium)iPS 細胞 .................................................... 21
3-2-3 UtE(human uterine endometrium)iPS 細胞 ........................................... 22
3-3 幹細胞特殊生物標記之鑑定 ......................................................................... 23
3-3-1 mES 多功能性因子 Sox2 & Oct3/4 之表現 ........................................ 23
3-3-2 mES 核蛋白標的雙重染色Oct3/4-Nanog............................................ 24
3-3-3 人類幹細胞專一性抗體SSEA4&TRA-1-60 ........................................ 25
3-3-4 人類多功能性因子 Sox2 & Oct3/4 之表現 ......................................... 27
3-3-5 人類核蛋白標的雙重染色Oct3/4-Nanog ............................................ 29
第四章討論 ....................................................................................................................... 31
4-1 小鼠胚胎幹細胞的建立 ................................................................................. 31
4-2 人類誘導多能性幹細胞培養 ......................................................................... 32
4-3 小鼠胚胎幹細胞及人類誘導多能性幹細胞鑑定 .......................................... 33
第五章總結 ....................................................................................................................... 35
第六章參考文獻 ............................................................................................................... 36

參考文獻

第六章參考文獻
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指導教授

凌慶東(Qing-Dong Ling)

審核日期

2010-7-15

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