本文選題：CHIR　切入點：Wnta　出處：《安徽醫(yī)科大學學報》2017年01期

【摘要】：目的觀察Wnt/β-catenin信號通路激活劑CHIR99021和Wnt3a在小鼠胚胎干細胞(m ESCs)向心肌細胞分化中的作用。方法采用懸浮一步培養(yǎng)法形成擬胚體(EBs),在EBs形成后第2天至第5天分別加入CHIR99021、Wnt3a,命名為CHIR99021組及Wnt3a組,將自動分化的EBs作為對照組。用免疫熒光法檢測心肌特異性蛋白肌球蛋白重鏈(α-MHC)、肌鈣蛋白T(cTNT)及膜聯(lián)蛋白43(CX43)的表達。用qRT-PCR檢測中胚層標志基因Brachyury、心肌前體細胞標志基因Nkx2.5、心肌細胞特異性標志基因α-MHC、cTnT和Cx43 mRNA的表達。用Western blot法檢測α-MHC、cTNT和CX43蛋白表達。結(jié)果免疫熒光法提示各組m ESCs能夠向心肌細胞分化。在CHIR99021和Wnt3a誘導分化過程中,Brachyury在分化第7天達高峰;Nkx2.5、α-MHC、cTnT和Cx43的mRNA表達量隨著分化時間的延長而逐漸增加,第15天增加最為明顯,且CHIR99021組和Wnt3a組高于對照組;CHIR99021組優(yōu)于Wnt3a組(P0.05,P0.01)。Western blot檢測結(jié)果顯示CHIR99021組和Wnt3a組α-MHC、cTNT和CX43蛋白表達量明顯高于對照組,CHIR99021組高于Wnt3a組。結(jié)論 CHIR99021和Wnt3a在分化早期階段通過活化Wnt/β-catenin信號通路可促進m ESCs心肌細胞分化,且CHIR99021促心肌分化作用優(yōu)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 (偽 -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 偽 -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 偽 -MHCcTNT and CX43 protein was detected by Western blot method. On the 7th day of differentiation, the mRNA expression of Nkx2.5, 偽 -MHCcTnT and Cx43 increased with the prolongation of differentiation time. The most significant increase was on the 15th day, The expression of 偽 -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 偽 -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.
【作者單位】： 安徽醫(yī)科大學第一附屬醫(yī)院心血管內(nèi)科;
【基金】：安徽高校自然科學研究項目(編號:KJ2015ZD24)
【分類號】：R542.22

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