目的探索并验证原代大鼠肾小球系膜细胞最佳电转染条件。方法设立不同的电压与电容组合、电击前孵育温度、质粒DNA浓度等电转染条件,将绿色荧光蛋白表达质粒pEGFP-C1导入原代大鼠肾小球系膜细胞。24h后比较各组细胞存活率和绿色荧光蛋白表达情况,确立最佳电转染条件。再以此条件电转染质粒pcDNA3.1和pcDNA3.1-FHL2,48h后收取两组细胞RNA和蛋白。应用RT-PCR和Western印迹检测目的基因FHL2的核酸和蛋白表达,验证电转染效果。结果在电击前室温孵育,40μg质粒,340V电压、550μF电容的最佳电转染条件下,原代大鼠肾小球系膜细胞的电转染效率可达40%以上,细胞存活率在50%左右。转染FHL2质粒组较对照组检测FHL2的核酸和蛋白表达水平明显升高(P〈0.05)。结论适当的电转染条件可以有效转染原代大鼠肾小球系膜细胞。
Objective To study and validate the effective electric transfection conditions in primary glomerular mesangial cells of rats.Methods Twenty-four hours after green fluorescent protein(GFP) expression plasmid pEGFP-C1 was transfected into primary glomerular mesangial cells of rats under different electric pressures and capacities,incubation temperature,and plasmid DNA concentration,survival of cells and expression of green fluorescent protein were compared to determine the optical electric transfection conditions.Forty-eight hours after pcDNA3.1 and pcDNA3.1-FHL2 were transfected into the same cells under these conditions,RNA and protein were harvested.Expression of nucleic acid and protein in target gene were detected by RT-PCR and Western blot to validate the efficiency of electric transfection.Results The incubation temperature,40μg of plasmid,340V and 550μF were found to be the optimal electric transfection conditions in primary glomerular mesangial cells of rats with an electric transfection rate of above 40% and a cell survival rate of about 50%.The expression level of nucleic acid and protein was obviously higher in FHL2-transfected plasmid group than in control group(P0.05).Conclusion Primary mesangial cells of rats can be effectively transfected under appropriate electric transfection conditions.