博碩士論文 972211011 詳細資訊




以作者查詢圖書館館藏 以作者查詢臺灣博碩士 以作者查詢全國書目 勘誤回報 、線上人數:17 、訪客IP:35.173.234.140
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)
相關論文
★ 發展酵素非限制性全基因體調控因子解析方法★ 大腸癌細胞株之 EGFR—K-ras 訊號路徑的基因微陣列實驗 與化學基因體學分析
★ 由神經生長因子誘導之細胞內訊號路徑活化的化學基因體學分析★ 細胞週期蛋白D1 mRNA在小鼠胚胎及成體幹細胞和腫瘤細胞中的表現及其受多能性相關因子影響之探討
★ 運用時間序列微陣列資料來預測調控基因★ 以大鼠嗜鉻性瘤細胞株建立神經訊號傳遞之細胞分子生物學模型
★ 運用高通量基因微矩陣列方法解析由嗜鉻 細胞分化成神經細胞之全基因體的調控★ 神經生長因子在神經分化中轉錄因子活性及基因調控機制之橫觀
★ 以CRSBP-1接合子調控巨噬細胞的移動及吞噬★ Chemogenomic and Molecular Analysis of Signal Transduction Pathways in In Vivo and In Vitro Models
★ 探討人類子宮內膜 L-selectin ligands 在月經週期的表現
檔案 [Endnote RIS 格式]    [Bibtex 格式]    [相關文章]   [文章引用]   [完整記錄]   [館藏目錄]   至系統瀏覽論文 ( 永不開放)
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
1. Wobus, A.M. and K.R. Boheler, Embryonic stem cells: prospects for developmental biology and cell therapy. Physiol Rev, 2005. 85(2): p. 635-78.
2. 游正博、錢宗良等編著, 幹細胞學. 教育部顧問室 幹細胞與組織工程教學資源中心. 民國九十七年二月.
3. HENRY E. YOUNG, et al., Adult Stem Cells. WILEY-LISS 2004. 276A: p. 75-102.
4. Jiang, Y., et al., Pluripotency of mesenchymal stem cells derived from adult marrow. Nature, 2002. 418(6893): p. 41-9.
5. Takahashi, K. and S. Yamanaka, Induction of pluripotent stem cells from mouse
embryonic and adult fibroblast cultures by defined factors. Cell, 2006. 126(4): p. 663-76.
6. LJ Kliensmith, G.P., Multipotentiality of single embryonal carcinoma cells. Cancer Res
24, 1964: p. 1544–1552.
7. Andrews, P.W., et al., Embryonic stem (ES) cells and embryonal carcinoma (EC) cells: opposite sides of the same coin. Biochem Soc Trans, 2005. 33(Pt 6): p. 1526-30.
8. Chan, A.W., et al., Clonal propagation of primate offspring by embryo splitting.
Science, 2000. 287(5451): p. 317-9.
9. Temple, S., Embryonic stem cell self-renewal, analyzed. Cell, 2003. 115(3): p. 247-8.
10. Martin, M.J., et al., Human embryonic stem cells express an immunogenic nonhuman sialic acid. Nat Med, 2005. 11(2): p. 228-32.
11. Evans, M.J. and M.H. Kaufman, Establishment in culture of pluripotential cells from mouse embryos. nature, 1981. 292(5819): p. 154-6.
12. Martin, G.R., Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A, 1981. 78(12): p. 7634-8.
13. Yu, J., et al., Induced pluripotent stem cell lines derived from human somatic cells. science, 2007. 318(5858): p. 1917-20.
14. Odorico, J.S., D.S. Kaufman, and J.A. Thomson, Multilineage differentiation from
human embryonic stem cell lines. Stem Cells, 2001. 19(3): p. 193-204.
15. Thomson, J.A., et al., Embryonic stem cell lines derived from human blastocysts. science, 1998. 282(5391): p. 1145-7.
16. Takahashi, K., et al., Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 2007. 131(5): p. 861-72.
17. Takahashi, K., et al., Induction of pluripotent stem cells from fibroblast cultures. Nat Protoc, 2007. 2(12): p. 3081-9.
18. 林培正副教授等編著, 科普化教材—人類胚胎幹細胞的研究與應用. 國科會科教處, 2005. 中山醫學大學醫學檢驗暨生物技術學系.
19. Lijun Yang, et al, In vitro trans-differentiation of adult hepatic stem cells into
pancreatic endocrine hormone-producing cells. PNAS June 11, 2002 99 p. 8078-8083
20. Oh, S.H., et al., Adult bone marrow-derived cells trans-differentiating into
insulin-producing cells for the treatment of type I diabetes. Lab Invest, 2004. 84(5): p.
607-17.
21. Pittenger, M.F., et al., Multilineage potential of adult human mesenchymal stem cells. science, 1999. 284(5411): p. 143-7.
22. 劉華昌、錢宗良等編著, 再生醫學. 中華民國九十七年二月.
23. Cartwright, P., et al., LIF/STAT3 controls ES cell self-renewal and pluripotency by a Myc-dependent mechanism. Development, 2005. 132(5): p. 885-96.
24. Woltjen, K., et al., piggyBac transposition reprograms fibroblasts to induced
pluripotent stem cells. nature, 2009. 458(7239): p. 766-70.
25. Charles A. Goldthwaite, J., Ph.D. , The promIse of Induced pluripotent stem cells(ipscs). Stem cell information NIH, 2010.
26. Ko K and Scholer HR., Embryonic stem cells as a potential source of gametes. .
Seminar of Reproductive Medicine 24, 2006. .
27. Cedar, S.H., et al., From embryos to embryonic stem cells: biopolitics and therapeutic potential. Reprod Biomed Online, 2006. 13(5): p. 725-31.
28. Damjanov, I., The road from teratocarcinoma to human embryonic stem cells. Stem Cell Rev, 2005. 1(3): p. 273-6.
29. Semb, H., Human embryonic stem cells: origin, properties and applications. APMIS, 2005. 113(11-12): p. 743-50.
30. Chambers, S.M., et al., Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol, 2009. 27(3): p. 275-80.
31. CONGER, K., Dramatic transformation: Researchers directly turn mouse skin cells into neurons, skipping IPS stage. Stanford school of medicine, 2010
32. Bleil, J.D. and P.M. Wassarman, Synthesis of zona pellucida proteins by denuded and follicle-enclosed mouse oocytes during culture in vitro. Proc Natl Acad Sci U S A, 1980. 77(2): p. 1029-33.
33. Svalander, P.C., et al., Trophectoderm surface expression of the cell adhesion molecule cell-CAM 105 on rat blastocysts. Development, 1987. 100(4): p. 653-60.
34. Dani, C., et al., Paracrine induction of stem cell renewal by LIF-deficient cells: a new ES cell regulatory pathway. Dev Biol, 1998. 203(1): p. 149-62.
35. Jeong, C.H., et al., Hypoxia-inducible factor-1 alpha inhibits self-renewal of mouse embryonic stem cells in Vitro via negative regulation of the leukemia inhibitory factor-STAT3 pathway. J Biol Chem, 2007. 282(18): p. 13672-9.
36. Mo, C., W. Chearwae, and J.J. Bright, PPARgamma regulates LIF-induced growth and self-renewal of mouse ES cells through Tyk2-Stat3 pathway. Cell Signal, 2010. 22(3): p. 495-500.
37. Bommhardt, U., et al., MEK activity regulates negative selection of immature
CD4+CD8+ thymocytes. J Immunol, 2000. 164(5): p. 2326-37.
38. Xu, C., et al., Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol, 2001. 19(10): p. 971-4.
39. Lunyak, V.V. and M.G. Rosenfeld, Epigenetic regulation of stem cell fate. Hum Mol Genet, 2008. 17(R1): p. R28-36.
40. Avilion, A.A., et al., Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Dev, 2003. 17(1): p. 126-40.
41. Masui, S., et al., Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells. Nat Cell Biol, 2007. 9(6): p. 625-35.
42. Makino, H., et al., Mesenchymal to embryonic incomplete transition of human cells by chimeric OCT4/3 (POU5F1) with physiological co-activator EWS. Exp Cell Res, 2009. 315(16): p. 2727-40.
43. Looijenga, L.H., et al., POU5F1 (OCT3/4) identifies cells with pluripotent potential in human germ cell tumors. Cancer Res, 2003. 63(9): p. 2244-50.
44. Loh, Y.H., et al., The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat Genet, 2006. 38(4): p. 431-40.
45. Cavaleri, F. and H.R. Scholer, Nanog: a new recruit to the embryonic stem cell
orchestra. Cell, 2003. 113(5): p. 551-2.
46. Faherty, S., et al., Self-renewal and differentiation of mouse embryonic stem cells as measured by Oct4 expression: the role of the cAMP/PKA pathway. In Vitro Cell Dev Biol Anim, 2007. 43(1): p. 37-47.
47. Aleksander Giwercman, et al., lmmunohistochemical Expression of Embryonal Marker TRA-I-60 in Carcinoma In Situ and Germ Cell Tumors of the Testis CANCER 1993. 72 p. 1308-1314.
48. Chambers, I., et al., Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell, 2003. 113(5): p. 643-55.
49. Henderson, J.K., et al., Preimplantation human embryos and embryonic stem cells show comparable expression of stage-specific embryonic antigens. Stem Cells, 2002. 20(4): p. 329-37.
50. Levenstein, M.E., et al., Basic fibroblast growth factor support of human embryonic stem cell self-renewal. Stem Cells, 2006. 24(3): p. 568-74.
51. Draper, J.S., et al., Surface antigens of human embryonic stem cells: changes upon differentiation in culture. J Anat, 2002. 200(Pt 3): p. 249-58.
52. Nagata, S., et al., Efficient reprogramming of human and mouse primary
extra-embryonic cells to pluripotent stem cells. Genes Cells, 2009. 14(12): p. 1395-404.
指導教授 凌慶東(Qing-Dong Ling) 審核日期 2010-7-15
推文 facebook   plurk   twitter   funp   google   live   udn   HD   myshare   reddit   netvibes   friend   youpush   delicious   baidu   
網路書籤 Google bookmarks   del.icio.us   hemidemi   myshare   

若有論文相關問題,請聯絡國立中央大學圖書館推廣服務組 TEL:(03)422-7151轉57407,或E-mail聯絡  - 隱私權政策聲明