CHIR99021和Wnt3a在小鼠胚胎干细胞向心肌细胞分化中的作用  

Effects of CHIR99021 and Wnt3a on cardiac differentiation of mouse embryonic stem cells

作　　者：邹传德 杨谋广 王爱玲[1] 

机构地区：[1]安徽医科大学第一附属医院心血管内科,合肥230022

出　　处：《安徽医科大学学报》2017年第1期73-78,共6页Acta Universitatis Medicinalis Anhui

基　　金：安徽高校自然科学研究项目(编号:KJ2015ZD24)

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(P<0.05,P<0.01)。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。Objective To investigate the effects of CHIR99021 and Wnt3a, Wnt/β-catenin signaling pathway activators, on cardiac differentiation of mouse embryonic stem cells （mESCs）. Methods The embryonic bodies （EBs） were formed through suspension culture method, CHIR99021 or Wnt3a was added into differentiated medium from day 2 to 5, named CHIR99021 group or Wnt3a group, respectively. In addition, there was a control group in which EBs were automatically differentiated. The expression levels of Brachyury, the mesoderm specific target gene, and Nkx2.5, cardiac-precursor marker, as well as the transcripts of eardiomyocyte markers, α-myosin heavy chain （a-MHC）, cardiac troponin T （cTnT） and eonnexin-43 （Cx43） were analyzed through quantitative RT- PCR. Besides, the cardiac-specific proteins including a-MHC, cTNT and CX43 were detected by immunofluores- cence and Western blot. Results The mESCs in every group did differentiate into eardiomyocytes. The expression of Brachyury was substantially augmented by treatment with CHIR99021 and Wnt3a, showing a peak of expression at day 7. Similarly, CHIR99021 and Wnt3a dramatically increased the expression levels of Nkx2.5, c-MHC, cT- nT and Cx43 with the time of differentiation, with the expression of target genes in CHIR99021 group and Wnt3a group was greater than that in the control group and CHIR99021 group was higher than Wnt3a group at day 15 （P 〈0. 05, P 〈 0. 01 ）. Western blot analysis suggested that the expressions of cL-MHC, cTNT and CX43 in CHIR99021 group and Wnt3a group were greater than those in the control group, and CHIR99021 group was higher than Wnt3a group at day 15. Conclusion Both CHIR99021 and Wnt3a could improve cardiogenesis from mESCs through activate Wnt/β-catenin signaling pathway at the early stage of differentiation while the former is better than the latter.

关 键 词：CHIR99021 WNT3A 小鼠胚胎干细胞 心肌细胞 

分 类 号：R349.7[医药卫生—基础医学]