单核细胞化学吸引蛋白质1 小分子干扰RNA人肾小管上皮细胞株的建立

目的 建立单核细胞化学吸引蛋白质1（MCP-1） 小分子干扰RNA（siRNA）人肾小管上皮细胞株，并检测MCP-1 siRNA对人肾小管上皮细胞（HKC）MCP-1基因的抑制效应。 方法 针对人MCP-1 mRNA 67、116、142位点设计并合成3对MCP-1 siRNA序列。构建MCP-1特异性siRNA真核表达载体，以脂质体法瞬时转染至HKC。转染24 h后分别应用实时定量RT-PCR及Western印迹技术检测HKC内MCP-1 mRNA、蛋白表达，筛选出抑制效率最高的MCP-1 siRNA。以筛选出的MCP-1 siRNA序列构建慢病毒穿梭质粒。使用慢病毒穿梭质粒进行慢病毒颗粒的包装和生产，得到病毒液并确定其滴度。以病毒液感染HKC，筛选MCP-1 siRNA人肾小管上皮细胞株，并应用实时定量RT-PCR及Western印迹技术检测HKC内MCP-1 mRNA、蛋白表达。 结果 成功建立MCP-1特异性siRNA人肾小管上皮细胞株。MCP-1 siRNA稳定转染能下调人肾小管上皮细胞MCP-1 mRNA (68.49±6.38)%、蛋白(72.97±6.13)%，与对照组比较差异有统计学意义(P < 0.01)。 结论 MCP-1特异性siRNA能高效抑制HKC内MCP-1基因表达。MCP-1 siRNA人肾小管上皮细胞株的建立为MCP-1基因功能及肾间质纤维化防治研究提供实验材料和依据。

Establishment of monocyte chemoattractant protein-1 small interfering RNA human renal tubular epithelial cells line

Objective To establish MCP-1 siRNA human renal tubular epithelial cells line and to investigate the inhibitory effect of plasma-mediated small interfering RNA (siRNA) on the expression of monocyte chemoattractant 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 specific for MCP-1 was constructed and transiently transfected into HKC by lipofectamine. At 24 hour after transfection, the mRNA and protein expression of MCP-1 was detected by real time RT-PCR and Western blot, respectively. The most effective sequence and device lentivaris plasmids were chosen and lentivaris granule’s packaging as well as production were applied. Virus fluid was derived and its density was identified. HKC were transfected by virus fluid and MCP-1 siRNA human renal tubular epithelial cells line was established. The mRNA and protein expression of MCP-1 was detected by real time RT-PCR and Western blot, respectively. Results MCP-1 siRNA human renal tubular epithelial cells line were successfully established . Compared with the normal control group, the mRNA and protein expression of MCP-1 siRNA stable transfection group were markedly down-regulated [(68.49±6.38)%,（72.97±6.13）%, respectively] （P<0.01）. Conclusions MCP-1 siRNA can highly inhibit the expression level of MCP-1 gene. It suggests MCP-1 siRNA human renal tubular epithelial cells line be crucial for offering experimental data to detect gene function and prevent renal interstitial fibrosis.