SMAD2介导的Activin A信号对人胚胎干细胞向限定性内胚层细胞诱导分化的促进作用

SMAD2 mediates Activin signaling in promoting definitive endoderm differentiation of human embryonic stem cells

【目的】研究SMAD2在Activin A促进人胚胎干细胞向限定性内胚层诱导分化中的作用。【方法】在人胚胎干细胞中转染SMAD2基因siRNA或者过表达质粒后,收集经Activin A分别诱导0,6,12,24,48,72,96和120h的细胞各100ng/mL,采用实时定量PCR检测SMAD2与内中胚层共同前体标记Brachyury和内胚层标记Sox17的表达,进一步通过Western-blot分析Activin A诱导中SMAD2和磷酸化SMAD2(p-SMAD2)表达的变化。【结果】在Activin A诱导人胚胎干细胞向限定性内胚层细胞分化的过程中,干扰SMAD2后48h时才检测到Brachyury强表达,而单纯Activin A处理组24h就检测到强表达;Sox17的表达始终较单纯Activin A处理组明显降低,因此,干扰SMAD2直接抑制了Activin A的诱导作用。而过表达SMAD2,Brachyury和Sox17的表达水平较单纯Activin A处理组明显增加,促进了限定性内胚层的发生;并且在Activin A诱导过程中,p-SMAD2的表达水平明显提高,而SMAD2的表达没有明显改变。【结论】SMAD2作为关键因子,介导了Activin A诱导人胚胎干细胞向限定性内胚层的分化,并转录调控Brachyury和Sox17的表达;SMAD2的磷酸化,激活并介导了Activin A诱导的信号转导通路。

【Objective】 The study was conducted to confirm the affection of SMAD2 by which Activin A promoted the definitive endoderm（DE） differentiation from human embryonic stem cells（hESCs）.【Method】 hESCs were transfected with SMAD2 siRNA or expression plasmids,and were detected with the mesoendoderm precursor related gene Brachyury and endodermal gene Sox17 expression changes by real-time quantitative PCR on 0,6,12,24,48,72,96 and 120 h respectively,with the treatment of 100 ng/mL Activin A.Furthermore,Western-blot analysis was carried out to detect the SMAD2 and p-SMAD2 expression levels.【Result】 In the process of Activin A induction in DE differentiation of hESCs,Brachyury expression was significantly increased after 24 h in Activin A simply treated group,while it was significantly increased after 48 h with SMAD2 interference.SMAD2 RNAi inhibited the process of Activin A induction in DE differentiation.Compared with the Brachyury and Sox17 expressions in Activin A treated group,SMAD2 overexpression promoted the Brachyury and Sox17 genes expressions in the process of Activin A induction in DE differentiation of hESCs.With Activin A induction,the p-SMAD2 expression levels were also upregulated.【Conclusion】 These data indicate that SMAD2 is a key regulator of Activin A induction in DE differentiation of hESCs,which regulates developmental events through SMAD2 transcriptional modulators and promotes Brachyury and Sox17 expression.In the process of Activin A induction,p-SMAD2 expression was upregulated to promote the transcription of target genes of Activin A induction.