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標題: | 探討小鼠胚胎幹細胞與誘導式多能幹細胞之神經分化差異性 Study of Differences in Neural Differentiation between Mouse Embryonic Stem Cells and Induced Pluripotent Stem Cells |
作者: | Wen-Chin Huang 黃文勤 |
指導教授: | 錢宗良 |
關鍵字: | 胚胎幹細胞,誘導式多能幹細胞,神經分化,維他命A酸,胞外信號調節激酶, embryonic stem cell,induced pluripotent stem cell,neural differentiation,retinoic acid,Erk1/2, |
出版年 : | 2011 |
學位: | 碩士 |
摘要: | 胚胎幹細胞是一群來自於囊胚期內細胞團的多能性幹細胞,擁有自我更新及分化成三胚層所有細胞的能力。而誘導式多能幹細胞是利用四個轉錄因子Oct4、Sox2、Klf4及c-Myc轉染到體細胞中,藉由再程序化作用使體細胞回到類似胚胎幹細胞狀態的幹細胞。誘導式多能幹細胞由於是取自於體細胞,因此在發育學研究、藥物篩選與自體細胞移植具有比胚胎幹細胞更好的應用優勢。
在此篇研究中,我們比較了小鼠胚胎幹細胞與誘導式多能幹細胞的神經分化潛力。在體外分化的試驗中,我們利用神經誘導分子“維他命A酸”去誘導胚胎幹細胞與誘導式多能幹細胞分化成神經幹細胞/前驅細胞、神經細胞、星狀膠細胞及寡突膠細胞。根據流式細胞儀、及時定量聚合酶連鎖反應之分析結果,我們可以發現在胚胎幹細胞與誘導式多能幹細胞的早期神經分化基因的表現上具有顯著的差異,此外,再利用免疫細胞化學染色去觀察早期與晚期神經分化的表現,我們可以發現小鼠胚胎幹細胞與誘導式多能幹細胞幾乎皆有表現早期與晚期神經分化標記,但是在表現量上面都具有明顯的差異。為了知道維他命A酸及其下游之訊息傳導機制是否會造成神經分化之差異性,我們利用即時定量聚合酶連鎖反應去分析維他命A酸受器與維他命A酸代謝相關酵素及其下游纖維母細胞生長因子之基因表現,再利用西方點墨法去觀察胞外信號調節激酶之磷酸化表現量。在此訊息傳遞的表現分析上,小鼠胚胎幹細胞與誘導式多能幹細胞幾乎都達到統計上的顯著差異,而且胚胎幹細胞的表現量皆大於誘導式多能幹細胞。 在此篇研究中,我們可以觀察到不論在早期或是晚期神經誘導分化,胚胎幹細胞之神經分化潛力皆優於誘導式多能幹細胞。而這兩種幹細胞對於維他命A酸的反應的不同可能是造成維他命A酸及其下游之訊息傳遞表現量差異的原因,因而產生不同的神經分化能力。另外,維他命A酸及其下游之纖維母細胞生長因子與胞外信號調節激酶訊息傳遞在神經分化中可能扮演重要的角色。 Embryonic stem cells (ESCs) possess powerful ability to self-renew and to differentiate into all cell types of three germ layers. It has been reported that induced pluripotent stem cells (iPSCs) derived from somatic cells have been generated by transfecting four transcription factors including Oct4, Sox2, Klf4 and c-Myc. iPSCs provide advantages in various applications, such as developmental studies, pharmaceutical screening, and autologous cell transplantation. iPSCs have also showed powerful ability of self-renewal and differentiation into a variety of cell types and resemble the properties of ESCs. In this study, we compared the neural differentiation potency of mouse ESCs with that of iPSCs. We demonstrated the mouse ESCs and iPSCs generated neural stem/progenitor cells and various neural lineage cells in vitro with the neural inducer “retinoic acid (RA)” treatment. However, the differential expression of early neural genes between ESCs and iPSCs was observed by flow cytometry and quantitative PCR. Different developmental patterns were also observed during early and late stages of neural differentiation via immunocytochemistry. To test whether RA-Fgf/Erk pathway caused the differences in neural induction of ESCs and iPSCs, we used quantitative PCR to analyze gene expression of RA receptors and RA-metabolizing enzymes and performed Western blotting to assess the phosphorylation level of Erk1/2. We found that the RA receptors, RA-metabolizing enzymes and phosphorylation of Erk1/2 were expressed at significantly different levels between ESCs and iPSCs. Our results suggest that the neural differentiation potency of ESCs was observed higher than that of iPSCs, and the RA-Fgf/Erk pathway may play an important role in neural differentiation between ESCs and iPSCs. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/32974 |
全文授權: | 有償授權 |
顯示於系所單位: | 解剖學暨細胞生物學科所 |
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