小鼠胚胎干细胞分化心肌细胞血管紧张素Ⅱ 1型和2型受体的表达特征

Expression of Angiotensin Ⅱ Type 1 and Type 2 Receptors in Mouse Embryonic Stem Cell-derived Cardiomyocytes

胚胎干细胞(ESC)具有无限增殖和分化为体内3个胚层来源的各种类型组织细胞的潜能,经过体外诱导能够分化为心肌细胞,亦称为胚胎干细胞分化心肌细胞(ESCM).本研究探讨了ESC诱导分化心肌细胞过程中血管紧张素Ⅱ受体(ATR)的亚型AT1R和AT2R的表达特征.10-4mol/L维生素C体外诱导小鼠R1胚胎干细胞分化为自发搏动的心肌细胞,用免疫荧光法检测分化后的细胞表达心肌细胞特异性标志物α辅肌动蛋白.小鼠胚胎干细胞在诱导分化为心肌细胞以后,逆转录聚合酶链反应(RT-PCR)和实时定量RT-PCR(Real-timeRT-PCR)方法检测到ESCM表达AT1R,并且呈时间依赖性逐渐增加的特点,在第14d达到高峰.Western印记法检测AT1R表达特征与RT-PCR结果相符.Western印记法的结果显示,血管紧张素Ⅱ(10-6mol/L)可作为AT1R激动剂激活AT1R,并使其下游的细胞外信号调节激酶(ERK1/2)磷酸化水平上调,预孵育AT1R抑制剂Losartan(10-6mol/L),此作用被抑制.RT-PCR方法显示,与新生小鼠心室肌细胞相比,ESCM的AT2R表达水平较低.

Embryonic stem cells（ESC） have the capacity for multilineage differentiation,including cardiomyocytes.Cardiomyocytes derived from embryonic stem cells（ESCM） have been well-investigated,but the developmental process of myocardial cells has not been systematically studied yet,due to the lack of a good study model.There are three functional type receptors on myocardial cell surface,adrenoceptors,muscarinic receptors and angiotensin Ⅱ receptors.Our group has revealed the expression profile of adrenoceptors and muscarinic receptors in ESCM previously.In the present study,we investigated the expression of angiotensin Ⅱ type 1 receptor（AT1R） and type 2 receptor（AT2R） during the differentiation process of ESCM.The R1 mouse ESC were induced by 10-4 mol /L vitamin C to differentiate into spontaneous beating cardiomyocytes.The expression of cardiac specific marker α-actinin was detected using immunofluorescent technique in differentiated ESCM.Real-time RT-PCR and Western-blotting results revealed that the expression of AT1R increased in a time dependent manner during the process of ESCM differentiation and reached the maximal level at differentiation day 14.An exogenous AT1R agonist,angiotensin Ⅱ（10-6 mol/L）,could activate AT1R and caused phosphorylation of extracellular responsive kinase（ERK1/2） in ESCM.This process could be inhibited by an AT1R antagonist,Losartan（10 -6 mol/L）.RT-PCR showed that AT2R expressed at a low level,compared to neonatal mouse cardiomyocytes,during the process of mouse.