Mas基因沉默对血管紧张素-(1-7)拮抗血管紧张素Ⅱ诱导的大鼠肾间质成纤维细胞活化的影响

摘要

AIM:To explore the influence of Mas gene silencing on the effect of angiotensin-(1-7) [Ang-(1-7)] that antagonizes rat renal interstitial fibroblast activation induced by angiotensin Ⅱ (Ang Ⅱ).METHODS:(1) To select the most effective Mas siRNA,three different sequences of Mas siRNA were designed and synthesized according to the sequence of Mas gene,and transiently transfected into rat renal interstitial fibroblasts (NRK-49F cells).The cells were divided into 5 groups:Mas siRNA-1 group,Mas siRNA-2 group,Mas siRNA-3 group,negative siRNA group and normal control group.The mRNA and protein expression of Mas was detected by RT-PCR and Western blotting 48 h after transfection,respectively.(2) To investigate the influence of the selected Mas siRNA on the antagonizing effect of Ang-(1-7) on Ang Ⅱ,NRK-49F cells were divided into6 groups:normal control group,AngⅡ group,Ang-(1-7) group,AngⅡ +Ang-(1-7) group,Ang Ⅱ + Ang-(1-7) + negative siRNA group and Ang Ⅱ + Ang-(1-7) + Mas siRNA group.The cells were cultured for 72 h,and then the expression of α-smooth muscle αctin (α-SMA) was detected by the method of immunocytochemistry.The content of collagen type Ⅰ (Col Ⅰ) in the culture supernatant was measured by ELISA.RESULTS:Compared with negative siRNA group,Mas siRNAs could effectively inhibit the expression of Mas both at mRNA and protein levels,and Mas siRNA-2 was the most effective (P ＜ 0.05).After treatment with Ang Ⅱ and Ang-(1-7) for 72 h,the expression of α-SMA and the secretion of Col Ⅰ in the cells of Mas siRNA group were significantly increased compared with non-transfection group and negative siRNA group (P ＜ 0.05).CONCLUSION:Mas siRNA inhibits the expression of Mas and then eliminates the antagonizing effect of Ang-(1-7) on rt renal interstitial fibroblast activation induced by Ang Ⅱ.%目的:探讨Mas基因沉默后对血管紧张素-(1-7)[Ang-(1-7)]拮抗血管紧张素Ⅱ(AngⅡ)诱导的大鼠肾间质成纤维细胞活化的影响.方法:(1)有效Mas siRNA的筛选:设计合成3对不同序列针对Mas基因的siRNA,瞬时转染大鼠肾间质成纤维细胞(NRK-49F),实验分5组:Mas siRNA序列1、Mas siRNA序列2、Mas siRNA序列3、阴性siRNA序列及正常对照组.转染后48 h,分别用半定量RT-PCR及Western blotting检测Mas mRNA和蛋白的表达水平.(2)研究有效Mas siRNA对Ang-(1-7)拮抗AngⅡ的影响:实验分6组:正常对照组、AngⅡ组、Ang-(1-7)组、AngⅡ+Ang-(1-7)组、阴性siRNA对照组和Mas siRNA转染组.分别培养72 h后,细胞免疫化学法检测细胞活化标志物α-平滑肌肌动蛋白(α-SMA)的表达,ELISA法检测上清液中细胞外基质成分Ⅰ型胶原(ColⅠ)的含量.结果:(1)与组相比,Mas siRNA各组Mas基因的表达均下降,其中以Mas siRNA序列2最明显(P＜0.05).(2)有效Mas siRNA转染组在加AngⅡ+Ang-(1-7)干预72 h后,较非转染组和阴性siRNA转染组α-SMA及Col Ⅰ表达均增加,差异有统计学意义(P＜0.05).结论:Mas siRNA能有效抑制Mas的表达,使Ang-(1-7)拮抗AngⅡ诱导的大鼠肾间质成纤维细胞活化作用明显减弱.