Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/24875
標題: 利用嵌合小鼠模式探討重新表現Oct-4與Nanog之3T3纖維母細胞分化多能性
Analysis of the pluripotency of 3T3 firborblasts re-expressed Oct-4 and Nanog in chimeric mice
作者: 謝賢修
Shie, Shian-Shiou
關鍵字: chimeric mice;嵌合小鼠;Oct-4;Nanog;Oct-4;Nanog
出版社: 動物科學系所
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摘要: 
胚胎幹細胞(embryonic stem cells, ES cells)為一具有分化多能性(pluripotency)之細胞,可自我複製、分化成各種細胞,進而發育成不同組織與器官。ES cells多能性的維持與一些轉錄因子的表現與活性有關,其中Oct-4 與Nanog 為重要調控因子之一,其可維持ES cells 之不分化狀態與自我更新。因此,本研究之主要目的
為利用以CMV 啟動子啟始Oct4 與Nanog 之表現質體(分別稱之為CMV-Oct4 與CMV-Nanog)共轉染至表現綠螢光蛋白質(enhanced green fluorescent protein,EGFP)小鼠成纖維細胞株﹙EGFP-3T3 cell line﹚中,並利用嵌合動物(chimeric animals)之產製,以瞭解Oct-4 與Nanog 在分化體細胞再程式化(reprogramming)作用中之影響。於試驗一中將已構築好之CMV-Oct4 與CMV-Nanog 質體共轉染至EGFP-3T3 細胞中,並以G418 篩選6-7 代後,再利用反轉錄聚合酶連鎖反應(reverse transcription polymerase chain reaction, RT-PCR)分析其基因表現。結果顯示,共轉染之EGFP-3T3 細胞(稱之為EGFP-3T3-CMV 細胞)其Oct-4 與Nanog 之mRNA
可重新表現。試驗二利用細胞注射法(cell injection)分別將EGFP- ES cells、EGFP-3T3 與EGFP-3T3-CMV 細胞注入小鼠囊胚中,經胚移置後,比較三者之出生率、懷孕率與子代嵌合情形。結果顯示,三者之出生率與懷孕率均無顯著差異,但只有EGFP-ES cells 組能成功產下嵌合子代。試驗三,將三組細胞分別注入小鼠
囊胚後並進行胚移置,於胚齡6.5 天時回收胚胎,進行免疫組織化學染色(immunohistochemistry)觀察外源性細胞於早期嵌合胚之分布情形。結果顯示,只有EGFP-ES cells 嵌合胚有綠螢光蛋白表現。本研究中未發現來自EGFP-3T3-CMV 細胞之嵌合動物,可能是由於Oct-4 與Nanog 之再表現,不足以使體細胞完全恢復其分化多能性,但仍需進一步之試驗分析,以瞭解此二轉錄因子對體細胞再程式化之影響,以及反分化(dedifferentiation)細胞於嵌合胚之分布情形。

The pluripotent embryonic stem cells (ES cells) are able to undergo self-renewal and differentiation into a variety of different cell types. It has been shown that the pluripotency of ES cells was maintained by the critical regulatory genes. Among which, two transcription factors, Oct4 and Nanog, are necessary for it. The aims of this study were to investigate the roles of Oct4 and Nanog in the reprogramming of somatic cells by the production of chimeric animals. In Experiment 1, 3T3 cell line expressing enhanced green fluorescent protein (EGFP-3T3) was co-transfected with Oct4 and Nanog expression vector driven by CMV promoter (designated as CMV-Oct4 and CMV-Nanog). After 6-7 subcultures selected by G418, the expressions of Oct-4 and Nanog in the co-transfected EGFP-3T3 cells (designated as EGFP-3T3-CMV cells) were analyzed by reverse transcription polymerase chain reaction (RT-PCR). Results showed that Oct-4 and Nanog were re-expressed in EGFP-3T3-CMV cells. In Experiment 2, the chimeric mouse blastocysts were produced by microinjection of EGFP-ES, EGFP-3T3 and EGFP-3T3-CMV cells into blastocoele, respectively. After embryo transfer, the percentages of pregnancy and birth rates among the three grouops showed no significant differences. However, compared to 14.7% of chimerism in EGFP-ES group, no chimeric animal was born in both EGFP-3T3 and EGFP-3T3-CMV groups. In Experiment 3, the E6.5 embryos were collected after chimeric blastocyst transfer. The distribution of EGFP cells in the chimeric fetuses was analyzed by immunohistochemistry. The results showed that only EGFP-ES cells could contribute to the fetus. In conclusion, the re-expression of Oct4 and Nanog might not be able to recover the pluripotency of somatic cells. However, further investigations are required for understanding the effects of Oct4 and Nanog on the reprogramming of somatic cells and the distribution of dedifferentiated cells in the chimeric embryos.
URI: http://hdl.handle.net/11455/24875
其他識別: U0005-2808200711380800
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