【目的】研究Activin A对人胚胎干细胞（hESCs）向限定性内胚层（DE）诱导分化的促进作用及其信号通路分子,为hESCs向DE诱导分化体系的优化提供参考。【方法】在人饲养层体系培养的hESCs中,收集100ng/mL Activin A分别诱导0,6,12,24,48,72,96,120h的细胞,用实时荧光定量RT-PCR检测原条标记Gsc和Mixl1、中内胚层共同前体标记Brachyury、内胚层标记Foxa2和Sox17、中胚层标记Flk1、外胚层基因Pax6、多能性相关基因Oct4与Nanog表达水平的变化,用细胞免疫荧光检测Brachyury和Sox17蛋白表达水平的变化。【结果】在人饲养层（HEF）培养体系上,高浓度Activin A能更快地促进中内胚层基因的表达并提高其表达水平;Brachyury和Sox17蛋白的细胞免疫荧光检测表明,Activin A诱导12和48h就可检测二者的表达明显增加,且二者的表达水平分别在诱导48和96h时达到高峰;hESCs高效分化为限定性内胚层细胞,DE细胞分化率为（81.7±5.4）%,并且体外的内胚层分化过程遵循从原条开始、经过中内胚层共同前体阶段、再到内胚层的发育过程,与体内发育规律相似。【结论】Activin A能特异性地诱导人胚胎干细胞向限定性内胚层分化,转录调控Brachyury和Sox17蛋白的表达。%【Objective】 The study was conducted to confirm the affection and the signaling pathway molecules of Activin in promotion of the definitive endoderm（DE） differentiation from human embryonic stem cells（hESCs）,which will provide reference for induction optimization of DE differentiation from hESC.【Method】 Cells were cultured on human embryonic fibroblast cells（HEF）.Real-time quantitative RT-PCR were preformed to detect the expression of prime streak related genes（Gsc,Mixl1）,mesoendoderm precursor related gene Brachyury,endodermal genes（Foxa2,Sox17）,mesodermal marker Flk1,ectoderm gene Pax6,pluripotent genes（Oct4,Nanog）.Brachyury and Sox17 protein expression was further confirmed by immunofluorescence staining on time 0,6,12,24,48,72,96 and 120 h respectively,with the treatment of 100 ng/mL Activin A.【Result】 After Activin A’s induction,the endoderm and mesoderm genes’ expressions were upregulated more quickly and dominantly.Immunofluorescence analysis showed that with Activin A’s induction,Brachyury and Sox17 proteins expression significantly increased after 12 and 48 h,and reached the top after 48 and 96 h,respectively,in which the DE ratio was up to（81.7±5.4）%.Futhermore,endoderm differentiation process in vitro followed from the prime streak,mesoendoderm precursor,then to the endoderm development process,which was similar to the vivo developmental law.【Conclusion】 The data indicate that Activin A induction in DE differentiation of hESCs can regulate developmental events through transcriptional modulators and promote Brachyury and Sox17 proteins expression.
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