E3泛素连接酶c-CBL对血管紧张素Ⅱ诱导后小鼠原代心肌成纤维细胞表型转换影响

Effects of E3 ubiquitin-protein ligase c-casitas B-lineage lymphoma on phenotypic transformation of rat primary cardiac fibroblasts after angiotensin Ⅱ stimulation

目的探讨E3泛素连接酶c-CBL对血管紧张素Ⅱ(AngⅡ)诱导后小鼠原代心肌成纤维细胞表型转换的影响。方法将细胞分为对照组(未刺激)与AngⅡ刺激组(不同浓度AngⅡ刺激细胞24 h),荧光定量聚合酶链式反应(PCR)和Western blot检测c-CBL、α-平滑肌肌动蛋白(αSMA)表达。应用小干扰RNA转染法建立低表达c-CBL细胞(si-CBL)后,将细胞分为si-control组(小干扰RNA对照转染24 h),si-CBL组(c-CBL小干扰RNA转染24 h),si-control+AngⅡ组(小干扰RNA对照转染24 h后给予AngⅡ刺激24 h)以及si-CBL+AngⅡ组(c-CBL小干扰RNA转染24 h后给予AngⅡ刺激24 h)。应用荧光定量PCR和Western blot检测各组细胞c-CBL、αSMA和胶原1(collagen 1)表达,应用免疫荧光染色检测各组细胞collagen 1表达。结果 AngⅡ组αSMA、c-CBL基因mRNA和蛋白均明显高于对照组,差异有统计学意义(P <0. 05)。siCBL组c-CBL基因mRNA及蛋白的表达显著低于si-control组,差异有统计学意义(P <0. 05)。AngⅡ刺激后,si-control+AngⅡ组和si-CBL+AngⅡ组αSMA基因mRNA、蛋白的表达均较si-control组和si-CBL组明显增加,si-CBL+AngⅡ组αSMA基因mRNA和蛋白均明显低于si-control+AngⅡ组,差异有统计学意义(P <0. 05)。AngⅡ刺激后,si-control+AngⅡ组和siCBL+AngⅡ组collagen 1基因mRNA均较si-control组和si-CBL组明显增加,且si-CBL+AngⅡ组collagen 1基因mRNA水平明显低于si-control+AngⅡ组,差异均有统计学意义(P <0. 05)。结论 c-CBL可影响AngⅡ诱导后小鼠原代心肌成纤维细胞的表型转换。

Objective To investigate the effect of E3 ubiquitin ligase c-casitas B-lineage lymphoma(c-CBL)on angiotensinⅡ(AngⅡ)induced mice after the original generation of myocardial fibroblasts phenotypic transition.Methods Cells can be divided into the control group(not stimulated),AngⅡstimulated group(different concentrations of AngⅡstimulates cell for 24 hours),fluorescence quantitative(polymerase chain reacion,PCR)and Western blot test c-CBL andαsmooth muscle actin(αSMA)expression.Application of small interfering RNA transfection technique to establish a lower expression c-CBL(si-CBL)cells,the cells into si-control group(small interference RNA contrast transfection for 24 hours),si-CBL group(c-CBL small interference RNA transfection for 24 hours),si-control+AngⅡgroup(small interference RNA controls transfection after 24 hours to give AngⅡstimulus for 24 hours)and si-CBL+AngⅡgroup(c-CBL small interference RNA transfection after 24 hours to give AngⅡstimulus for 24 hours).Application of fluorescence quantitative PCR and Western blot test each cell c-CBL,αSMA and collagen 1 expression,application of immunofluorescence staining detection collagen 1 groups of cells.Results TheαSMA、c-CBL gene mRNA and protein AngⅡgroup were significantly higher than the control group(P<0.05).The mRNA and protein expression levels of c-CBL gene in the si-CBL group were significantly lower than those in the si-control group(P<0.05).The mRNA and protein expression levels of c-CBL gene in the si-CBL group were significantly lower than those in the si-control group(P<0.05).AngⅡstimulation,si-control+AngⅡgroup and si-CBL+AngⅡαSMA gene mRNA and protein expression of si-control group and si-CBL group was obviously increased;αSMA gene mRNA and protein of si-CBL+AngⅡgroup were significantly lower than those of si-control+AngⅡgroup(P<0.05).After AngⅡstimulation,αSMA gene mRNA and protein expression of si-control+AngⅡgroup and si-CBL+AngⅡgroup were increased more obviously than those in the si-control group and si-CBL group;theαSMA gene mRNA and protein expression of si-control+AngⅡgroup and si-CBL+AngⅡof si-control group and si-CBL group was obviously increased;αSMA gene mRNA and protein of si-CBL+AngⅡgroup were significantly lower than those of si-control+AngⅡgroup(P<0.05).Conclusion The c-CBL could affect the phenotypic transformation of rat primary cardiac fibroblasts after AngⅡstimulation.