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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 TBX18 adenovirus vector transfected neonatal cardiomyocytes can induce its differentiation into pacemaker-like cells. Methods The neonatal rat cardiomyocytes were isolated and cultured by mixed digestion with trypsin and type Ⅱ collagenase. The cell morphology was observed under optical microscope. The purity of cardiomyocytes was detected by immunofluorescence after 48 hours. Cardiac myocytes were randomly divided into experimental group (TBX18 group) and empty virus control group (GFP group). Addition of adenovirus containing TBX18 transcription factor and green fluorescent protein (GFP), GFP-containing adenovirus, Fluorescent quantitative polymerase chain reaction, Western blot and immunofluorescence were used to detect the expression of HCN4 mRNA and HCN4 protein in each group. Results After cultured for 24h, cardiomyocytes almost completely adherent, showing fusiform, rhombic or polygonal; 48h after the formation of reticular or radial cell clusters. α-actin detection of myocardial cell purity up to 96%. The expression of HCN4 mRNA and HCN4 protein in TBX18 group was significantly higher than that in GFP group (28 943.997 ± 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). Under inverted fluorescence microscope, the expression of HCN4 protein in the TBX18 group showed red fluorescence and the GFP group showed almost no HCN4 protein expression. Conclusion TBX18 adenovirus vector transfected neonatal cardiomyocytes in vitro, cardiomyocytes can differentiate into pacemaker-like cells.