SMAD2介导Activin A信号促进人胚胎干细胞向限定性内胚层细胞诱导分化.docVIP

  SMAD2 介导 Activin A 信号促进人胚胎干 向限定性内胚层细胞诱导分化# 孙懿1,2，周静1,2，林戈1,2，卢光琇1,2** 5 10 15 20 25 30 35 40 （1. 中南大学生殖与干细胞研究所，长沙 410078； 2. 国家干细胞工程研究中心，长沙 410078） 摘要：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的作用。在人饲 养层体系培养的人胚胎干细胞中，收集 100 ng/ml Activin A 分别诱导 0、6 小时、12 小时、 1、2、3、4、5 天不同时间点的细胞，Western-blot 分析 SMAD2 和磷酸化 SMAD2（p-SMAD2） 的表达改变, 细胞转染 SMAD2 基因 siRNA 或者过表达质粒, 检测 SMAD2 表达改变对 Activin A 诱导内中胚层共同前体标记 Brachyury 和内胚层标记 Sox17 的表达影响。结果提示： Activin A 在诱导人胚胎干细胞向限定性内胚层诱导分化过程中 p-SMAD2 的表达水平的明显 提高。干扰 SMAD2 直接抑制了 Activin A 的诱导作用,过表达 SMAD2 促进了 DE 的发生研究。 提示 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分化，转录 调控 Brachyury 和 Sox17 的表达。 关键词：生物医学工程学；人胚胎干细胞；限定性内胚层；Activin A；SMAD2 中图分类号：Q291, Q813. 1 Smad2 mediates Activin signaling in definitive endoderm differentiation of human embryonic stem cells SUN Yi1,2, ZHOU Jin1,2, LIN Ge1,2, LU Guangxiu1,2 (1. Institute of Reproductive and Stem Cell Engineering , Central South University, ChangSha 410078; 2. National Research and Engineering Center of Human Stem Cells, ChangSha 410078) Abstract: In this paper, the study was done to confirm the affection of SMAD2 by which Activin A promoted the definitive endoderm (DE) differentiation from human embryonic stem cells (hESCs). Western-blot analysis was carried out to detect the SMAD2 and p-SMAD2 expression levels on days 0, 6h, 12h, 1, 2, 3, 4 and 5 respectively, with the treatment of 100 ng/ml Activin A. Furthermore, hESCs treated with Activin A were detected with the mesoendoderm precursor related gene Brachyury and endodermal genes Sox17 genes expression changes after transfected with SMAD2 siRNA or expression plasmids. These results demonstrated that after Activin A’s induction, Brachyury and Sox17 genes expressions were upregulated more quickly and dominantly. Coherent with these, the p-SMAD2 expression levels were also upregulated. SMAD RNAi or overexpression inhibited or promoted the induction of Activin A. These data indicates that SMAD2 is a key regulator of Activin A induction in DE differenti