| 其他摘要 | 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. |
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