CHIR99021和Wnt3a在小鼠胚胎干细胞向心肌细胞分化中的作用

发布时间：2018-03-27 23:32

  本文选题：CHIR　切入点：Wnta　出处：《安徽医科大学学报》2017年01期

【摘要】：目的观察Wnt/β-catenin信号通路激活剂CHIR99021和Wnt3a在小鼠胚胎干细胞(m ESCs)向心肌细胞分化中的作用。方法采用悬浮一步培养法形成拟胚体(EBs),在EBs形成后第2天至第5天分别加入CHIR99021、Wnt3a,命名为CHIR99021组及Wnt3a组,将自动分化的EBs作为对照组。用免疫荧光法检测心肌特异性蛋白肌球蛋白重链(α-MHC)、肌钙蛋白T(cTNT)及膜联蛋白43(CX43)的表达。用qRT-PCR检测中胚层标志基因Brachyury、心肌前体细胞标志基因Nkx2.5、心肌细胞特异性标志基因α-MHC、cTnT和Cx43 mRNA的表达。用Western blot法检测α-MHC、cTNT和CX43蛋白表达。结果免疫荧光法提示各组m ESCs能够向心肌细胞分化。在CHIR99021和Wnt3a诱导分化过程中,Brachyury在分化第7天达高峰;Nkx2.5、α-MHC、cTnT和Cx43的mRNA表达量随着分化时间的延长而逐渐增加,第15天增加最为明显,且CHIR99021组和Wnt3a组高于对照组;CHIR99021组优于Wnt3a组(P0.05,P0.01)。Western blot检测结果显示CHIR99021组和Wnt3a组α-MHC、cTNT和CX43蛋白表达量明显高于对照组,CHIR99021组高于Wnt3a组。结论 CHIR99021和Wnt3a在分化早期阶段通过活化Wnt/β-catenin信号通路可促进m ESCs心肌细胞分化,且CHIR99021促心肌分化作用优于Wnt3a。
[Abstract]:Objective to investigate the role of Wnt/ 尾 -catenin signal pathway activators (CHIR99021 and Wnt3a) in the differentiation of mouse embryonic stem cells into cardiomyocytes. Methods Wnt/ 尾 -catenin signaling activators (CHIR99021 and Wnt3a) were added to Wnt/ 尾 -catenin signaling pathway in vitro from day 2 to day 5 after the formation of EBs. CHIR99021 Wnt3a, named CHIR99021 group and Wnt3a group, The expression of myocardial specific myosin heavy chain (伪 -MHCC) and troponin 43 CX43 were detected by immunofluorescence. The mesodermal marker gene Brachyuryand the myocardial precursor marker were detected by qRT-PCR. The expression of 伪 -MHCC TnT and Cx43 mRNA was detected by Western blot. Results Immunofluorescence assay showed that m ESCs could differentiate into cardiomyocytes. During the process of CHIR99021 and Wnt3a induced differentiation, the expression of 伪 -MHCcTNT and CX43 protein was detected by Western blot method. On the 7th day of differentiation, the mRNA expression of Nkx2.5, 伪 -MHCcTnT and Cx43 increased with the prolongation of differentiation time. The most significant increase was on the 15th day, The expression of 伪 -MHCC TNT and CX43 in CHIR99021 group and Wnt3a group was significantly higher than that in control group CHIR99021. Conclusion CHIR99021 and Wnt3a can activate Wnt/ 尾 -catenin in the early stage of differentiation by the activation of Wnt/ 尾 -catenin at the early stage of differentiation. The results show that the expression of 伪 -MHCC TNT and CX43 in CHIR99021 group and Wnt3a group is significantly higher than that in control group CHIR99021 group. Signal pathway can promote the differentiation of m ESCs cardiomyocytes. The effect of CHIR99021 on myocardial differentiation was superior to that of Wnt3a.
【作者单位】： 安徽医科大学第一附属医院心血管内科;
【基金】：安徽高校自然科学研究项目(编号:KJ2015ZD24)
【分类号】：R542.22

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