转录因子Tbx18对升主动脉平滑肌细胞的重编程作用  

Role of transcription factor Tbx18 in reprogramming ascending aortic smooth muscle cells

作　　者：王凤媛 唐艳红[1] 王晞[1] 赵庆彦[1] 王腾[1] 黄从新[1] Wang Fengyuan;Tang Yanhong;Wang Xi;Zhao Qingyan;Wang Teng;Huang Congxin(Department of Cardiology,Wuhan University People's Hospital,Wuhan 430060,Hubei Province,China)

机构地区：[1]武汉大学人民医院心内科武汉大学心血管病研究所心血管病湖北省重点实验室

出　　处：《中华老年心脑血管病杂志》2019年第12期1309-1313,共5页Chinese Journal of Geriatric Heart,Brain and Vessel Diseases

基　　金：湖北省技术创新专项（2016ACA153）;中央高校基本科研业务费专项资金（2042015kf0229）

摘　　要：目的探讨转录因子Tbx18腺病毒载体在体外转染升主动脉平滑肌细胞的重编程作用。方法采用组织块贴壁法培养升主动脉平滑肌细胞,4~7d后在光学显微镜下观察细胞形态,细胞传代后随机分为空白组、绿色荧光蛋白(GFP)组、Tbx18组,Tbx18组转染携带Tbx18和GFP腺病毒,GFP组转染等量GFP腺病毒,空白组不转染病毒。转染4d后通过RT-qPCR和Western blotting检测3组相关转录因子环核苷酸门控离子通道蛋白亚型4(HCN4)、Tbx3、NKx2.5和心肌细胞特异性标志物心肌肌钙蛋白Ⅰ(cTnⅠ)表达;通过免疫荧光技术检测Tbx18组及GFP组HCN4蛋白表达。结果与GFP组和空白组比较,Tbx18组HCN4、Tbx3和cTnⅠ mRNA和蛋白表达明显上调,NKx2.5蛋白表达明显降低,差异有统计学意义(0.135±0.033 vs 0.291±0.104、0.344±0.067,P<0.05)。免疫荧光结果显示,倒置荧光显微镜下Tbx18组HCN4蛋白发出红色荧光,并且与细胞转染的绿色荧光和细胞核蓝色荧光大致重合,而GFP组几乎无红色荧光发出。结论 Tbx18腺病毒载体在体外能够将升主动脉平滑肌细胞重编程为起搏样细胞。Objective To study the role of transcription factor Tbx18 in reprogramming in vitrotransfected ascending aortic SMC.Methods After the ascending aortic SMC were cultured by tissue block adherence for 4-7 days,their morphology was observed under light microscope.The SMC were randomly divided into blank group,GFP group and Tbx18 group after they were passaged.The SMC in Tbx18 group were transfected with adenovirus carrying transcription factor Tbx18 and GFP and those in GFP group were transfected with an equal amount of adenovirus carrying GFP while those in blank group were not transfected with adenovirus.The expressions of transcription factors HCN4,Tbx3,NKx2.5 and cardiomyocyte specificity marker-cTnⅠ in 3 groups were detected by RT-qPCR and Western blotting respectively while those of HCN4 protein in Tbx18 group and GFP group were detected by immunofluorescence.Results The expression levels of HCN4,Tbx3 and cTnⅠ were significantly higher while those of NKx2.5 protein were significantly lower(0.135±0.033 vs0.291±0.104,0.344±0.067,P<0.05)in Tbx18 group than in GFP group and blank group.Immunofluorescence showed red HCN4 protein fluorescence in Tbx18 group overlapped with SMC-transfected green fluorescence and nucleus blue fluorescence,and almost no red HCN4 protein fluorescence in GFP group.Conclusion Tbx18 adenoviral vector can reprogram the ascending aortic SMC into pacemaker-like cells in vitro.

关 键 词：转录因子 腺病毒科 主动脉 肌细胞 平滑肌 转染 肌钙蛋白Ⅰ 

分 类 号：R54[医药卫生—心血管疾病]