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目的比较早、晚代小鼠胚胎成纤维细胞(Mouse embryonic fibroblasts,MEF)维持人胚胎干细胞(Humanembryonic stem cells,h ESCs)正常生长的能力差异,并从分泌因子角度探讨其机制,为开发基于晚代MEF的h ESCs培养体系奠定基础。方法以P3或P9代MEF为饲养层培养h ESCs;通过添加碱性成纤维细胞生长因子(Basic fibroblastgrowth factor,b FGF)调节MEF分泌因子表达;用β-半乳糖苷酶活性染色检测细胞衰老;用定量RT-PCR检测基因的m RNA表达水平;用ELISA检测TNF-a的分泌量。结果 P3代MEF增殖较快,能维持h ESCs正常增殖,而P9代MEF呈现衰老表型,不能维持h ESCs正常增殖。P9代MEF表达多种分泌因子(Grem1、Tgfb1、Inhba、Bmp4和Tgfb2)及衰老相关分泌表型(Senescence-associated secretory phenotype,SASP)因子(Il-1a和Tnfa)的水平均明显高于P3代MEF。加入b FGF后,P3和P9代MEF的Grem1、Tgfb1和Inhba的表达均明显升高,而Bmp4和Tgfb2表达均下降。结论 P9代MEF保留对b FGF的反应性,但有明显衰老表型及SASP,SASP的出现可能是P9代MEF不能维持h ESCs正常生长的重要原因。
Objective To compare the ability of mouse embryonic fibroblasts (MEFs) to maintain the normal growth of human embryonic stem cells (hESCs) in early and later generations, and to explore its mechanism from the perspective of secretion factors, MEF h ESCs culture system to lay the foundation. Methods The hESCs were cultured with P3 or P9 MEF. The expression of MEF secretion factor was regulated by adding basic fibroblast growth factor (b FGF). The cell senescence was detected by β-galactosidase activity staining. The mRNA expression of m RNA was detected by quantitative RT-PCR. The secretion of TNF-a was detected by ELISA. Results The P3 generation MEF proliferated rapidly and maintained the normal proliferation of hESCs. However, MEF showed a phenotype of aging in P9 and could not maintain the normal proliferation of hESCs. The levels of many secreting factors (Grem1, Tgfb1, Inhba, Bmp4 and Tgfb2) and Senescence-associated secretory phenotype (SASP) (Il-1a and Tnfa) in P9 generation MEF were significantly higher than those in P3 generation MEF. After addition of bFGF, the expression of Grem1, Tgfb1 and Inhba in P3 and P9 MEFs were significantly increased, while the expression of Bmp4 and Tgfb2 decreased. Conclusion The P9 generation MEF retains the reactivity to b FGF. However, the obvious aging phenotype and the appearance of SASP and SASP may be the important reasons that MEF can not maintain the normal growth of hCs in P9 generation.