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目的以RNA干扰技术沉默TGF-β诱导早期基因(TIEG)的表达,观察TIEG沉默对糖基化终末产物(AGEs)介导的肾小管上皮细胞Smad2表达的影响。方法以pshRNA-copGFP-lentivector作为载体,构建含TIEG特异siRNA的慢病毒载体psiRNA-TIEG,将其转染至大鼠近端肾小管上皮细胞(NRK52E),然后给予糖基化修饰的牛血清白蛋白(AGE-BSA)刺激24h和48h,采用PCR方法测定其TIEG mRNA、Smad2 mRNA表达水平,蛋白免疫印迹法测定Smad2蛋白表达水平。以转染空质粒载体组作为对照。结果TIEG特异的siRNA可有效下调AGEs诱导的TIEG mRNA、Smad2 mRNA和蛋白表达,与空质粒载体组相比差异有统计学意义(P<0.05)。结论TIEG的沉默能有效抑制AGEs介导的NRK52E细胞Smad2 mRNA和蛋白的表达。
Objective To silence the expression of early gene (TIEG) induced by TGF-β by RNA interference and observe the effect of TIEG silencing on the expression of Smad2 in glomerular mesangial cells mediated by advanced glycation end products (AGEs). Methods The lentiviral vector psiRNA-TIEG containing TIEG specific siRNA was constructed by using pshRNA-copGFP-lentivector as a vector and transfected into proximal tubular epithelial cells (NRK52E) in rats. Then, glycosylated bovine serum albumin (AGE-BSA) for 24h and 48h. The expression of TIEG mRNA and Smad2 mRNA was detected by PCR. The expression of Smad2 protein was detected by Western blotting. The empty plasmid vector group was used as a control. Results TIEG-specific siRNA effectively down-regulated AGEs-induced mRNA and protein expression of TIEG mRNA and Smad2, which was significantly different from that of empty plasmid vector group (P <0.05). Conclusion TIEG silencing can effectively inhibit AGEs-mediated Smad2 mRNA and protein expression in NRK52E cells.