不同密度大鼠成纤维细胞对心肌细胞Cx43及细胞活力的影响

Effects of different density rat fibroblasts on expression of Cx43 in cardiomyocytes and cell viability

目的评价不同密度大鼠成纤维细胞对心肌细胞缝隙连接蛋白43(Cx43)及细胞活力的影响。方法将心肌细胞与成纤维细胞进行Transwell小室共培养,心肌细胞接种于Transwell小室下层,成纤维细胞分别接种于Transwell小室上层,采用随机数字表法分为3组(n=12):成纤维细胞密度0.5×10^(5)个/ml组(C_(0.5)组)、成纤维细胞密度为1×10^(5)个/ml组(C_(1)组)和成纤维细胞密度为2×10^(5)个/ml组(C_(2)组),3组心肌细胞密度均为1×10^(5)个/ml。3组均共培养20 h。培养结束后,收集心肌细胞,采用CCK8法检测细胞活力,采用流式细胞术检测凋亡率,采用实时定量PCR法检测Cx43 mRNA表达,采用Western blot法检测Cx43及磷酸化(p-Cx43)表达。结果3组心肌细胞凋亡率比较差异无统计学意义(P>0.05)。与C_(0.5)组比较,C_(1)组心肌细胞Cx43及其mRNA、p-Cx43表达上调,C_(2)组心肌细胞活力升高,Cx43及其mRNA、p-Cx43表达上调(P<0.05);与C_(1)组比较,C_(2)组心肌细胞活力升高,Cx43及其mRNA、p-Cx43表达上调(P<0.05)。结论大鼠成纤维细胞在一定范围内呈密度依赖性上调心肌细胞Cx43表达和活性,增强细胞活力。

Objective To evaluate the effects of different density rat fibroblasts on the expression of conjunctin 43(Cx43)in cardiomyocytes and cell viability.Methods Cardiomyocytes and fibroblasts were co-cultured using Transwell,cardiomyocytes were inoculated into the lower chamber of Transwell and fibroblasts into the upper chamber of Transwell.The cells were divided into 3 groups(n=12 each)by a random number table method:fibroblast density 0.5×10^(5) cells/ml group(group C_(0.5)),fibroblast density 1×10^(5) cells/ml group(group C_(1)),and fibroblast density 2×10^(5) cells/ml group(group C_(2)),with the density of cardiomyocytes 1×10^(5) cells/ml in three groups.Cardiomyocytes and fibroblasts were co-cultured for 20 h in three groups.Cardiomyocytes were collected after co-culture for determination of cell viability(by CCK8 method),apoptosis rate(by flow cytometry),and expression of Cx43 mRNA(by quantitative real-time polymerase chain reaction)and expression of Cx43 and phosphorylated Cx43(p-Cx43)(by Western blot).Results There was no significant difference in the apoptosis rate of cardiomyocytes among the three groups(P>0.05).Compared with group C_(0.5),the expression of Cx43 protein and mRNA and p-Cx43 was significantly up-regulated in group C_(1),the cardiomyocyte viability was significantly increased,and the expression of Cx43 protein and mRNA and p-Cx43 was up-regulated in group C_(2)(P<0.05).Compared with group C_(1),the cardiomyocyte viability was significantly increased,and the expression of Cx43 protein and mRNA and p-Cx43 was up-regulated in group C_(2)(P<0.05).Conclusion Rat fibroblasts up-regulate the expression of Cx43 and enhance the activity of Cx43 in cardiomyocytes and enhance cell viability in a density-dependent manner in a certain range.