饲养层FGF2表达量对猕猴胚胎干细胞自我更新及多能性的影响

Influence of Different FGF2-expressing Feeder Layers on the Self-renewal and Pluripotency of Rhesus Monkey Embryonic Stem Cells

用转基因和RNA干扰(RNAi)法建立5组不同成纤维细胞生长因子-2(fibroblast growth factor-2,FGF2)表达量的猕猴耳部皮肤成纤维细胞(MESF)系:过表达FGF2组(f1),过表达的阴性对照组(f2),FGF2RNA干扰组(f3),RNA干扰的阴性对照组(f4)和未作任何处理的对照组(f5)。5组MESF的FGF2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FGF2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长期培养(10代)猕猴胚胎干细胞(RhESC),结果在f1上培养的RhESC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RhESC增殖较慢,BMP4表达上调,c-fos,TGF-β1,INHBA,Gremlin1,Oct-4,Nanog,Sox2表达下调。5组MESF上培养的RhESC形成的EB均表达各胚层早期标记基因(marker),说明RhESC的多能性没有受到影响,但表达量有差异,f1上RhESC形成的EB所有marker都低表达。结果表明饲养层的FGF2含量不仅影响自身相关基因的表达,还对RhESC的自我更新有一定的作用。

Transgene and RNAi were used to establish five different FGF2-expressing monkey ear skin fibroblast （MESF） cell lines, the FGF2 over-expressed line （f1）, the negative control of fl （f2）, RNA interfered line （f3）, the negative control of f3 （f4） and the non-treated control （f5）. The expression ratio of FGF2 in these lines was f1： f2： f3： f4： f5=4 ： 2 ：1 ： 2： 2. The results indicated that c-fos, TGF-β1, INHBA, Gremlinl were upregulated in fl but downregulated in f3, while BMP4, TGF-β2 were downregulated in fl but upregulated in f3, which implied that endogenous FGF2 affected the TGF-β signaling pathway and the expression level of related genes changed in MESFs. Further analysis of rhesus monkey embryonic stem cells （RhESCs） supported by these MESFs showed that RhESCs on fl grew more quickly than those in other groups, in which the expression level of c-fos, TGF-β1, INHBA, Gremlin1, OCT-4, Nanog and Sox2 was higher, while that of BMP4 was lower; in contrast, RhESCs on f3 grew more slowly than in other groups, in which the expression level ofc-fos, TGF-β1, INHBA, Gremlinl, OCT-4, Nanog and Sox2 declined, while that of BMP4 and TGF-β2 increased. EBs from these RhESCs expressed early markers representing all germ layers,but the expression levels of markers in RhESCs on fl were lower than in other groups. These findings demonstrated that different FGF2 expression in feeder layers can not only influence expression of relative genes in MESFs, but also affect proliferation and self-renewal of ESCs.