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题名不同FGF-2表达量的饲养层对猕猴和兔胚胎干细胞的增殖和自我更新的影响
作者张敬
学位类别硕士
答辩日期2008-03
授予单位中国科学院研究生院
授予地点北京
导师季维智
关键词FGF-2 饲养层 猕猴胚胎干细胞 兔胚胎干细胞 增殖 自我更新
其他题名Influence of different FGF-2 expression in feeder layers on monkey and rabbit embryonic stem cells proliferation and self-renewal
学位专业细胞生物学
中文摘要胚胎干细胞(ESC)培养是ESC研究的基础,饲养层的选择是ESC培养的一个重要方面。本实验曾对不同的猕猴饲养层进行研究,显示能够更好的支持猕猴ESC生长的饲养层FGF-2表达量也较高。FGF-2,又名bFGF(碱性成纤维生长因子),是ESC生长所需的重要因子,但其中的分子机制现在并未完全了解。本文一方面对ESC相关研究进展进行了综述,另一方面对以下内容进行了研究:用转基因和RNA干(RNAi)扰的方法建立不同FGF-2的表达量猕猴耳部皮肤细胞(MESF)系五组:过表达FGF-2(f1),过表达的阴性对照组(f2),FGF-2 RNA干扰组(f3),RNA干扰的阴性对照组(f4)以及未作任何处理的对照组(f5),这五组MESF的FGF-2表达量相对值为f1:f2:f3:f4:f5=4:2:1:2:2;c-fos,TGF-β1,INHBA,Gremlin1在f1中表达量上升,在f3中表达量下降;BMP4,TGF-β2在f1中表达量下降,在f3中表达量上升;表明内源FGF-2能够作用于MESF的TGF-β信号通路,引起相关基因表达量的变化。用这些细胞作为饲养层分别培养两种ESC(猕猴ESC,R366. 4和兔ESC,RFESC) ,连续培养了10代,其中在f1上培养的两种ESC增殖速度都比对照组快,并且c-fos,TGF-β1,INHBA,Gremlin1,OCT-4,Nanog,Sox2表达量均上升,BMP4表达下调;在f3上培养的猕猴ESC增殖较慢,BMP4表达上调,c-fos,TGF-β1,INHBA,Gremlin1,OCT-4,Nanog,Sox2表达下调;f3上的兔ESC没有变化。表明ESC中的TGF-β信号通路也受到调节。五组猕猴和兔的ESC形成的EB均表达各胚层早期标记基因(marker),但表达量有差异,f1上ESC形成的EB所有marker都低表达。实验结果表明饲养层中的FGF-2含量高低不仅影响自身相关基因的表达,还对ESC的增殖和维持自我更新有一定的作用。
英文摘要Feeder layer is important to culture of embryonic stem cells (ESC). Our previously study found that different monkey feeder layers had different abilities to support monkey ESC growth as FGF2 expression different, showed that among them the one which could support monkey ESC growth better were high FGF-2 expression. FGF-2 is an essential factor for ESC growth, but the molecular mechanism is still unclear. In this thesis, the progresses of ESC research have been reviewed, and the influence of FGF2 in feeder layer on monkey and rabbit ESC has been studied. Transgene and RNAi have been used to establish five different FGF-2 expression monkey ear skin fibroblast (MESF) cell lines: f1 was FGF-2 overexpression, f2 was negative control of f1, f3 was FGF-2 RNAi, f4 was negative control of f3, f5 was non-treated. FGF-2 relative quantities are f1: f2: f3: f4: f5=4: 2: 1: 2: 2. It was shown that endogenous FGF-2 could affect TGF-β signaling pathway in MESFs, and change some relative genes expression. We found that c-fos, TGF-β1, INHBA, Gremlin1 were upregulated in f1, while downregulated in f3, and BMP4, TGF-β2 were downregulated in f1, while upregulated in f3. It was also shown that TGF-β signaling pathway in ESC was regulated. Both monkey and rabbit ESCs were supported on these MESFs for 10 passages. ESCs on f1 growed more quickly, in which c-fos,TGF-β1,INHBA,Gremlin1,OCT-4,Nanog and Sox2 were expressed higher than other groups, while BMP4 were downregulated; oppositely, monkey ESCs on f3 growed more slowly than other groups, in which c-fos,TGF-β1,INHBA,Gremlin1,OCT-4,Nanog and Sox2 expressions declined, while BMP4 and TGF-β2 expression increased, but rabbit ESC on f3 was identical with control groups. EBs from these monkey and rabbit ESCs expressed early markers representative all three germ layers, but expression of markers in ESCs on f1 were lower than other groups. In conclusion, these findings showed that different FGF-2 expression in feeder layers can not only influence expression of relative genes in MESFs, but also affect proliferation and self-renewal of ESCs.
语种中文
公开日期2010-10-14
内容类型学位论文
源URL[http://159.226.149.42:8088/handle/152453/6094]  
专题昆明动物研究所_生殖与发育生物学
推荐引用方式
GB/T 7714
张敬. 不同FGF-2表达量的饲养层对猕猴和兔胚胎干细胞的增殖和自我更新的影响[D]. 北京. 中国科学院研究生院. 2008.
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