稳定敲低SIGIRR的人肾小管上皮细胞的构建及其初步功能的研究

摘要

Objective To establish single immunoglobin IL-1-related receptor ( SIGIRR) stable knockdown human renal tubular epithelial cells ( HKC) and explore its function. Methods Designed the effective targeted shRNA se-quences for the SIGIRR gene, and then connected with the GV248-GFP-Puro to produce recombinant lentiviral vec-tor ( GV248-GFP-Puro-shSIGIRR ) . The corrected recombinants, identified by sequenced, were transfected into 293T cells with packaging plasmids (pMDL, pRev, pVSVG) for virus packaging. HKC cells were then infected by packaged viruses. Quantitative real-time PCR( qRT-PCR) and Western blot were used to detect the interference effi-ciency of SIGIRR in HKC cells. SIGIRR stable knockdown HKC cells ( HKC/shSIGIRR ) and the control cells were induced by IL-1β, then the downstream nuclear transcription factor, the phosphorylated NF-κB ( p65 ) were detected by Western blot. The mRNA expression levels of chemokine monocyte chemotactic protein 1 (MCP-1) and regulated upon activation normal T cell expressed and secreted factor ( RANTES) were analyzed by qRT-PCR.Results The results showed that the recombinant lentiviral vector GV248-GFP-Puro-shSIGIRR was successfully constructed. qRT-PCR and Western blot confirmed that SIGIRR was knockdown in HKC cells. Moreover, the re-sults also showed that compared with the control cells, the phosphorylated NF-κB (p65) and the mRNA levels of MCP-1 and RANTES were significantly increased in HKC/shSIGIRR cells by being stimulated with IL-1β. Conclu-sion The results suggest that SIGIRR acts as a“brake” of inflammatory reaction in Toll-like receptor/IL-1 recep-tor (TLR/IL-1R) pathway in HKC cells. The study may provide a potential new target for the treatment of Lupus nephritis.%目的：建立稳定敲低单免疫球蛋白白介素1相关受体(SIGIRR)基因的人肾小管上皮细胞株(HKC),并初步研究其功能。方法针对SIGIRR基因的有效靶点设计shRNA序列,与 GV248-GFP-Puro 慢病毒载体连接产生重组体( GV248-GFP-Puro-shSIGIRR)。将测序验证正确的重组体与包装质粒(pMDL、pRev、pVSVG)共转染293T细胞进行病毒包装,收集病毒并感染HKC细胞。实时荧光定量PCR( qRT-PCR)和Western blot法分析检测HKC细胞中SIGIRR干涉效率。应用白细胞介素-1β( IL-1β)刺激成功敲低SIGIRR的HKC细胞及对照细胞, Western blot法检测其下游核转录因子NF-κB(p65)的磷酸化水平,qRT-PCR分析单核细胞趋化蛋白-1(MCP-1)、正常 T细胞表达和分泌的活化调节蛋白( RANTES) mRNA水平。结果成功构建重组慢病毒载体GV248-GFP-Puro-shSIGIRR。 qRT-PCR 和 Western blot 法均证实在HKC细胞中成功敲低SIGIRR的表达。此外, IL-1β刺激后,与对照细胞相比,敲低SIGIRR的HKC细胞( HKC/shSIGIRR)的p65磷酸化水平上调, MCP-1和 RANTES mR-NA表达水平升高。结论 HKC的炎症反应中,SIGIRR蛋白对Toll样受体/白介素1受体(TLR/IL-1R)通路起“刹车”作用。该研究为狼疮性肾炎的治疗提供了新的潜在的靶点。