早、晚代MEF用于人胚胎干细胞培养的能力差异及分泌机制

The capacity variance and secretory mechanisms of early and late generation of MEF in cultivation of human embryonic stem cells

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 capacity variance of early and late generation of MEF（Mouse embryonicfibroblasts）in supporting human embryonic stem cells（h ESCs）growth,and to investigate the mechanisms from the perspectiveof the secretion function,thus to provide the basis for creating new culture system of h ESCs based on the late generation ofMEF. Methods P3 and P9 generation of MEF were used to culture h ESCs. By adding b FGF（basic fibroblast growth factor）,the expression of excreted factors of MEF was regulated. Cell senescence was evaluated by β-galactosidase reactivity staining.Quantitative RT-PCR was employed to detect the m RNA expression level of genes and ELISA was used to detect the secretionlevel of TNF-a. Results P3 generation of MEF（P3 MEF）grew fast and could maintain the normal proliferation of h ESCs,while P9 MEF displayed senescence phenotype and unable to maintain normal proliferation of h ESCs. The expression level ofvarious genes which encoded secretory factors（Grem1,Tgfb1,Inhba,Bmp4 and Tgfb2）and senescence-associated secretoryphenotype（SASP）factors（Il1a,Tnfa）were significantly higher in P9 MEF than in P3 MEF. After the adding of basic fibroblastgrowth factor（b FGF）,the expression of Grem1,Tgfb1 and Inhba increased,while Bmp4 and Tgfb2 decreased in both P3 andP9 MEF. Conclusion The P9 MEF maintains the capacity of responding to b FGF,but the cells are senescence anddemonstrate SASP. The enhanced and aberrant secretion function of P9 MEF might be the reasons leading to its disability inh ESCs culture.