携带TBX18的慢病毒载体转染脂肪干细胞并诱导其向起搏样细胞分化的研究  

Transfection of adipose-derived stem cells with TBX18-carrying lentiviral vectors and inducing their differentiation into pacing-like cells

作　　者：王方嫒 邹强[1] 谌晶晶[1] 单迎光[1] 黄从新[1] WANG Fang-ai;ZOU Qiang;SHEN Jing-jing;SHAN Ying-guang;HUANG Cong-xin(Department of Cardiology, Renmin Hospital of Wuhan Universit;Cardiovascular Research Institute, Wuhan Universit;Hubei Key Laboratory of Cardiology, Wuhan 430060, Hubei,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,湖北武汉430060

出　　处：《中国心脏起搏与心电生理杂志》2018年第2期147-151,共5页Chinese Journal of Cardiac Pacing and Electrophysiology

基　　金：中央高校基本科研业务费专项资金(项目编号:2105kfD229)

摘　　要：目的观察TBX18慢病毒载体在体外转染脂肪干细胞(ADSCs),能否诱导其向起搏样细胞分化。方法取40~50g SD大鼠的腹股沟脂肪,采用酶混合消化法分离培养ADSCs,光学显微镜下观察细胞形态,将细胞随机分为TBX18组、GFP组、null组,TBX18组转染携带TBX18转录因子和绿色荧光蛋白(GFP)的TBX18慢病毒,GFP组转染等量的GFP慢病毒作为空病毒对照组,null组不转染病毒作为空白对照组。一周后通过蛋白质免疫印迹(Western blotting)检测三组相关转录因子TBX3、ISL1、SHOX2,缝隙连接蛋白CX45、CX43、CX30.2及心肌细胞特异性标志物心肌肌钙蛋白I(cTNI)、肌动蛋白(α-SMA)的蛋白表达量;通过免疫荧光技术检测TBX18组及GFP组的α-SMA蛋白的表达。病毒转染后第7、14、21、28天分别采用实时荧光定量聚合酶链式反(RT-PCR)检测TBX18组及GFP组TBX18及HCN4水平。结果 TBX18组转染ADSCs后,细胞形态发生改变,转染后4周内可通过荧光显微镜下观察到持续的荧光表达,诱导分化后,TBX18组TBX3、ISL1、SHOX2、CX45、CX30.2、cTN1、α-SMA蛋白水平明显高于GFP组及null组,而CX43蛋白表达低于null组;TBX18组免疫荧光检测到了α-SMA,而GFP组表达阴性;TBX18组TBX18及HCN4表达在慢病毒转染后4周内持续表达,明显高于GFP组(P<0.05)。结论 TBX18慢病毒载体体外转染入ADSCs,能在细胞内稳定表达,且能使其向起搏样细胞分化。Objective To observe whether TBX18 lentiviral vector is transfected into adipose-derived stem cells in vitro and can induce its differentiation into pacing-like cells.Methods The ADSCs were isolated and cultured by enzymatic digestion method from Sprague Dawley（SD）rat（40-50 g）.The cells were observed under light microscope.The cells were randomly divided into TBX18 group,GFP group and null group.The transfected cells were transfected with TBX18 transfection factor and green fluorescent protein（GFP）lentiviruses,the null group did not transfect the virus.One week later,the expression of related transcription factors TBX3,ISL1,SHOX2,connexin CX45,CX43,CX30.2 and cardiomyocyte specific markers cardiac troponin 1（cTN1）,actin（α-SMA）were detected by western bloting;andα-SMA protein expression in TBX18 group and GFP group was detected by immunofluorescence assay.The levels of TBX18 and HCN4 were detected by real-time fluorescence quantitative polymerase chain reaction（RT-PCR）on 7 th,14 th,21 th and 28 th day after transfection in TBX18 group and GFP group.Results After transfection of ADSCs in the experimental group（TBX18 group）,the morphology of the cells changed and the fluorescence expression was observed by fluorescence microscopy within 4 weeks after transfection.Western blotting showed that the levels of TBX3,ISL1,SHOX2,CX45,CX30.2,cTN1 and α-SMA were increased in the experimental group,while the expression of CX43 protein was lower than that of the null group.The immunofluorescence of the TBX18 group detectedα-SMA and the GFP group was negative.RT-PCR results showed that the expression of TBX18 and HCN4 in the experimental group was significantly higher than that in the control group at 4 weeks after lentivirus transfaction（P〈0.05）.Conclusion TBX18 lentiviral vector is transfected into adiposederived stem cells in vitro and can be expressed stably in cells,which can differentiate adipose-derived stem cells into pacing cells.

关 键 词：心血管病学 Tbx18 脂肪干细胞 慢病毒 诱导 分化 起搏细胞 生物起搏 

分 类 号：R318.11[医药卫生—生物医学工程]