摘要
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ColⅠ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。
AIM： To explore the influence of Mas gene silencing on the effect of angiotensin-（1-7） [Ang-（17） ]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 into 6 groups： normal control group,AngⅡ group,Ang-（1-7） group,AngⅡ ＋ Ang（1-7） group,AngⅡ ＋ Ang-（1-7） ＋ negative siRNA group and AngⅡ ＋ Ang-（1-7） ＋ Mas siRNA group.The cells were cultured for 72 h,and then the expression of α-smooth muscle actin（α-SMA） was detected by the method of immunocytochemistry.The content of collagen type Ⅰ（ColⅠ） 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 ColⅠin 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 rat renal interstitial fibroblast activation induced by Ang Ⅱ.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2013年第7期1225-1229,共5页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.30771008)
