TGF-βRⅡ siRNA对AngⅡ诱导GMC增殖的影响

Effect of TGF-βRⅡ siRNA on AngⅡ induced proliferation of glomerular mesangial cell

目的通过干扰TGF-βRⅡ的表达,探讨TGF-βRⅡ对AngⅡ诱导GMC增殖的作用及对TGF-β1与psmad3表达的影响。方法 GMC转入荧光对照siRNA,6 h于荧光显微镜观察沉默效率。分别转入阴性对照siRNA与TGF-βRⅡsiRNA,24 h Western blot检测TGF-βRⅡ的表达。实验分为:空白对照组(Con组),AngⅡ组、siRNA阴性对照组(NC组)、TGF-βRⅡsiRNA干扰组(沉默组)。除Con组外,AngⅡ刺激各组24 h,Western blot检验TGF-β1与psmad3的蛋白水平。CCK8试剂盒检测GMC增殖变化。结果 (1)转染TGF-βRⅡsiRNA后检测TGF-βRⅡ的蛋白水平表达明显降低,与对照组相比表达差异有统计学意义(P<0.05);(2)AngⅡ能刺激TGF-β1的表达,转染TGF-βRⅡsiRNA后,TGF-β1蛋白表达下降(P<0.05);(3)AngⅡ能促进smad3的磷酸化,转染TGF-βRⅡsiRNA后psmad3蛋白表达下降(P<0.05);(4)AngⅡ促进GMC增殖,转染TGF-βRⅡsiRNA后减轻AngⅡ的增殖作用(P<0.05)。结论 AngⅡ刺激细胞增殖与TGF-β1表达及smad3的磷酸化有关,且依赖TGF-βRⅡ的表达。

Objective To investigate the effect of transforming growth factorβreceptor-Ⅱ(TGF-βRⅡ)on AngⅡinducing glomerular mesangial cell(GMC)proliferation and the expressions of TGF-β1and psmad3through transfecting TGF-βRⅡsmall interfering RNA(siRNA)into GMC.Methods Through transfecting fluorescence control siRNA into GMC,we observed the transfection efficiency under fluorescence microscope after6hours.Transfecting TGF-βRⅡsiRNA and negative control siRNA into GMC respectively,we detected the expression of TGF-βRⅡby western blot after24hours.The cells were divided into four groups:control group,AngⅡgroup,negative siRNA control group and TGF-βRⅡsiRNA group.Each group was stimulated by AngⅡexcept the control group.After24hours,we detected the TGF-β1and psmad3protein levels by western blot and detected GMC proliferation by CCK8kit.Results(1)Comparing with the scrambled control group,the expression of TGFβRⅡin the TGF-βRⅡsiRNA group was significantly decreased(P<0.05).(2)AngⅡcould accelerate the expression of TGF-β1.Transfecting TGF?βRⅡsiRNA into GMC decreased the expression of TGF?β1protein(P<0.05).(3)AngⅡcould stimulate the phosphorylation of smad3.Transfecting TGF-βRⅡsiRNA into GMC decreased the expression of psmad3protein(P<0.05).(4)Transfecting TGF?βRⅡsiRNA into GMC relieved the GMC proliferation AngⅡ-promoted(P<0.05).Conclusions The AngⅡstimulates the GMC proliferation,depending on the expression of TGF?βRⅡ.It is related to the expressions of TGF-β1and psmad3.