GATA-4过表达增加大鼠骨髓间充质干细胞分化为心肌细胞  

Overexpressing GATA-4 increases rat bone marrow mesenchymal stem cells transdifferentiating into cardiomyocytes

作　　者：李红霞[1] 周亚峰[1] 杨向军[1] 蒋文平[1] 

机构地区：[1]苏州大学附属第一医院心内科,江苏苏州215006

出　　处：《基础医学与临床》2013年第1期60-65,共6页Basic and Clinical Medicine

基　　金：国家自然科学基金(81100173;81070139);江苏省普通高校研究生科研创新计划(CXZZ11_0118);江苏省医学创新团队与领军人才项目(LJ201140)

摘　　要：目的研究GATA-4转染骨髓间充质干细胞(MSCs)分化为心肌细胞及表达心肌细胞特异性标志物的能力。方法分离培养MSCs及心肌细胞(CM)。第3代MSCs转染GATA-4基因(MSCGATA-4)并通过实时定量聚合酶链反应(PCR)、Western blot和免疫组化鉴定GATA-4的表达。MSCGATA-4与CM嵌套式共培养1周,通过real-time PCR和Western blot检测其心肌基因BNP、α-actinin和Islet-1的表达,用免疫组化观察α-actinin的表达。与CM混合共培养后1周后,观察MSCs的搏动情况,并用流式细胞仪检测心肌分化率。结果 MSCGATA-4组GATA-4的表达明显高于对照组(只表达GFP)。MSC与CM嵌套式共培养后,MSCGATA-4组的BNP、α-actinin和Islet-1的mRNA和蛋白表达明显高于对照组(P<0.05)。混合共培养后,可见部分MSCGATA-4的搏动,同时部分MSC的α-actinin染色阳性。用流式细胞仪检测心肌分化率,MSCGATA-4组的心肌分化率明显高于对照组(P<0.05)。结论 GATA-4过表达可以增加MSCs分化成心肌样细胞,高表达BNP、α-actinin和Islet-1。Objective To investigate whether overexpression of GATA-4 increases adult mesenchymal stromal cell（MSC） transdifferentiation into a cardiac phenotype in vitro.Methods MSCs and cardiomyocytes（CMs） were isolated and cultured.MSCs were transduced with GATA-4（MSCGATA-4） at the third passage.Gene expression in MSCGATA-4 was analyzed using real-time PCR,Western blot and immunohistochemistry.After co-cultured with CM for one week,the expressions of cardiac genes of MSCGATA-4,including brain natriuretic peptide（BNP）,Islet-1 and α-sarcomeric actinin,were determined by RT-PCR and Western blot.After mixed coculture with CM for one week,the beating of MSC was observed.MSCs were examined for α-actinin by immunohistochemistry and the transdifferentiation rate of MSC was calculated directly from flow cytometery.Results Compared with control cells that were transfected with Green Fluorescent Protein（GFP） only（MSCNull）,MSCGATA-4 were shown by immunofluorescence,RT-PCR and Western blot to have higher expression of GATA-4.After incubation for one week,the mRNA and protein expression of cardiac genes,including BNP,Islet-1 and α-sarcomeric actinin,were up-regulated in MSCGATA-4 compared with MSCNull（P0.05）.After co-incubation for one week,some MSCs beated and some MSCs were positive for α-actinin staining.The transdifferentiation rate was signiflcantly higher in MSCGATA-4 than MSCNull（P0.05）.Conclusions Overexpression of GATA-4 signiflcantly increases MSC differentiation into a myocardial phenotype and expressing BNP,Islet-1 and α-actinin.

关 键 词：骨髓间充质干细胞 心肌细胞 GATA-4 共培养 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]