过表达C3aR人足细胞的构建及鉴定  

Construction and identification of a human podocytes strain over-expressing C3aR

作　　者：郑敬民[1] 尹广[1] 赵文紧[1] 刘志红[1] 

机构地区：[1]南京军区南京总医院国家肾脏疾病临床医学研究中心,全军肾脏病研究所,江苏南京210002

出　　处：《东南国防医药》2014年第6期561-565,共5页Military Medical Journal of Southeast China

基　　金：国家自然科学基金项目(81370828)

摘　　要：目的构建过表达C3a受体(C3aR)的肾小球足细胞株,为探讨C3aR在肾小球足细胞中的确切生理和病理意义提供细胞模型。方法设计合成人C3aR表达单元,将其克隆到慢病毒表达载体p Lenti6.3-MCS-IRES2-EGFP的多克隆位点,构建成C3aR表达载体p Lenti6.3-C3aR-IRES2-EGFP;将p Lenti6.3-C3aR-IRES2-EGFP和包装质粒共转染293 T细胞,包装成C3aR表达重组慢病毒LV-C3aR;以LV-C3aR感染人肾小球足细胞系HPC,根据LV-C3aR上带有杀稻瘟菌素抗性基因的特点,以杀稻瘟菌素筛选稳定转染细胞克隆;利用荧光定量PCR和细胞免疫化学分析方法对稳定转染细胞克隆的C3aR表达水平进行分析,从中鉴定出稳定过表达C3aR的人肾小球足细胞株。结果成功构建了C3aR表达载体p Lenti6.3-C3aR-IRES2-EGFP;得到了高滴度的C3aR表达重组慢病毒LV-C3aR;成功构建了过表达C3aR的人肾小球足细胞株HPC-C3aR。结论成功构建C3aR表达载体和过表达C3aR的人肾小球足细胞株,为进一步研究C3aR过表达在人肾小球足细胞中的病理意义提供了很好的细胞模型,也为进一步开展C3aR在其他细胞中的生理、病理意义创造了条件。Objective To investigate the function and pathological significance of C 3aR in the cells,in the present study,a podocytes strain over-expressing C3aR was constructed.Methods C3aR expression unit was designed ,synthesized and then cloned in-to the multi-clonal site of pLenti6.3-MCS-IRES2-EGFP,a lentivirus expression vector .After identification by sequencing ,recombinant lentivirus was packaged by using pLenti 6.3-C3aR-IRES2-EGFP and packaging plasmid in 293T cells.Then,the recombinant lentivirus was used to infect HPC ,a cell line of human glomerular podocytes .After screening in medium with blasticidin ,blasticidin resistant cell clones were obtained and then identified by real-time PCR and immunochemical staining for the expression of C 3aR.Results The C3aR expressing vector pLenti6.3-C3aR-IRES2-EGFP was successfully constructed;The recombinant lentivirus LV-C3aR was success-fully packaged;A Cell strain of glomerular podecytes over-expression C3aR was established.Conclusion The present study success-fully constructed a C3aR expression vector pLenti6.3-C3aR-IRES2-EGFP and a C3aR over-expression podocytes strain HPC-C3aR, which is very useful in further study of the function of C 3aR not only in renal tubular epithelial cells ,but also in other type of cells .

关 键 词：C3a受体 慢病毒 肾小球足细胞 过表达 

分 类 号：R363.1[医药卫生—病理学]