TBX18腺病毒载体体外诱导乳鼠心肌细胞向起搏样细胞分化

Direct conversion of neonatal rat ventricular myocytes to pacemaker cells by expression of TBX18in vitro

目的观察TBX18腺病毒载体在体外转染乳鼠心肌细胞,能否诱导其向起搏样细胞分化。方法胰酶和Ⅱ型胶原酶混合消化法分离培养乳鼠心肌细胞,光学显微镜下观察细胞形态,48h后用免疫荧光技术检测心肌细胞纯度。将心肌细胞随机分为实验组(TBX18组)、空病毒对照组(GFP组),转染时分别加入带TBX18转录因子和绿色荧光蛋白(GFP)的腺病毒,带GFP的腺病毒,采用实时荧光定量聚合酶链式反应,蛋白质免疫印迹和免疫荧光技术检测各组心肌细胞HCN4mRNA和HCN4蛋白的表达。结果培养24h后,心肌细胞几乎完全贴壁,可见梭形、菱形或多边形;48h后形成网状或放射状细胞簇。α-actin检测心肌细胞纯度高达96%。TBX18组心肌细胞中HCN4mRNA和HCN4蛋白的表达水平明显高于GFP组(28 943.997±5 019.682vs 1.000±0.000,n=9,P<0.05;0.631±0.025vs 0.192±0.003,n=9,P<0.05);倒置荧光显微镜下观察TBX18组因表达HCN4蛋白而发出红色荧光,GFP组几乎看不到HCN4蛋白的表达。结论 TBX18腺病毒载体体外转染乳鼠心肌细胞,能使乳鼠心肌细胞向起搏样细胞分化。

Objective To observe whether the adenoviral vectors expressing transcription factor gene TBX18 can induce neonatal rat ventricular myocytes（NRVMs）to sinoatrial node（SAN）-like pacemaker cells in vitro. Methods The NRVMs were digested and isolated by trypsin and typeⅡcollagenase,the basic shape of cardiomyocytes was observed under the opitical microscope.Immunofluoresccopy was used to detect the purity of NRVMs after 48 h.The cells were transfected with TBX18 and GFP,Quantitative Real-time PCR was used to detect the expression of HCN4 mRNA,Western blot,Immunofluoresccopy were used to detect the expression of protein of HCN4 channel. Results NRVMs were sticking to wall in 24 h,which expressing fusiformis,rhombus or polygon;after 48 h,the detached cells became connected into mesh or radial cell clusters.The purity of cardiomyocytes were up to 96%by the detection ofα-actin.The mRNA and protein expression levels of HCN4 in TBX18-infected cardiomyocytes were both markedly higher than those of the GFP-infected cardiomyocytes（28 943.99±5 019.682 vs 1.000±0.000,n=9,P〈0.05;0.631±0.025 vs 0.192±0.003,n=9,P 〈0.05）;TBX18-groups expressed red HCN4 protein,whereas GFP-groups can hardly see the expression of HCN4 protein under inverted fluorescence microscope. Conclusion TBX18 adenoviral vectors can induce the quiescent neonatal rat ventricular myocytes to SAN-like pacemaker cells in vitro.