质粒表达型小分子干扰RNA对人肾小管上皮细胞单核细胞趋化蛋白-1抑制作用的研究

Study on inhibition effect of small interfering RNA on monocyte chemotacite protein-1 in HKC

目的研究质粒表达型小分子干扰RNA(siRNA)对人肾小管上皮细胞(HKC)单核细胞趋化蛋白-1(MCP-1)的抑制作用。方法2002年2～7月对同济大学附属东方医院针对人MCP-1 mRNA67、116、142位点设计并合成的3对MCP-1siRNA序列,构建MCP-1特异性siRNA真核表达载体pRNAT-MCP-1-Ⅰ、pRNAT-MCP-1-Ⅱ、pRNAT-MCP-1-Ⅲ,以脂质体法转染至HKC,转染24h后分别采用实时定量反转录-聚合酶链反应(RT-PCR)及Western blot技术检测HKC内MCP-1 mRNA、蛋白表达。结果siRNA表达载体的转染效率为60%,与正常对照组相比,3组不同质粒表达型siRNAMCP-1 mRNA和蛋白表达均显著降低(P<0.01)。结论siRNA能高效抑制HKC的MCP-1基因表达,提示siRNA在防治肾间质纤维化中具有潜在作用。

Objective To investigate the inhibition effect of plasma-mediated small interfering RNA （siRNA） on the expression of monocyte chemotacite protein-1 （ MCP-1 ） gene in human renal tubular epithelial cells （ HKC ）. Methods Three pairs of siRNAs directed human＇s MCP-1 mRNA 67,116,142 targets were designed and synthesized. Eukaryotic expression vector special for MCP-1 ,pRNAT-MCP-1- Ⅰ ,pRNAT-MCP-1-Ⅱ ,pRNAT-MCP-1-Ⅱ were constructed and transfected into HKC by lipofectamine. At 24 hour after transfection,the expression of MCP-1 in the levels of mRNA was detected by Real Time RT-PCR, and the expression of MCP-1 in the levels of protein was detected by Western blot. Results Transfection efficiency of siRNA expression vector was 60% ;the expression of MCP-1 in the levels of mRNA and protein of three pairs of plasma-mediated siRNA group were markedly decreased compared with normal control group（ P 〈 0. 01 ）. Conclusion Plasma-mediated siRNA can highly inhibit the expression level of MCP-1 gene. It suggests plasma-mediated siRNA may be potential to prevent and treat renal interstitial fibrosis with excellent prospect of development.