过敏毒素C3a在肾小球足细胞中的分泌性表达试验  

Secrectory expression of anaphylotoxin C3a in a podocyte strain

作　　者：郑敬民[1] 尹广[1] 赵文紧[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》2015年第3期225-228,共4页Military Medical Journal of Southeast China

基　　金：国家自然科学基金项目(81370828)

摘　　要：目的建立过敏毒素C3a分泌性表达慢病毒体系和分泌性高表达过敏毒素C3a的足细胞株,为研究过敏毒素C3a对足细胞的作用创造条件。方法人工合成过敏素素C3a分泌性表达基因;以重组慢病毒为媒介,将过敏毒素C3a分泌性表达基因导入到人足细胞系(human podocytes,HPC);利用药物筛选和连续梯度稀释克隆法获得稳定整合有过敏毒素C3a转基因结构的HPC细胞株;利用实时定量PCR从mRNA水平、酶联免疫吸附测定方法从蛋白质水平对过敏毒素C3a的表达和分泌情况进行鉴定。结果成功建立了过敏毒素C3a分泌性表达慢病毒体系;获得了分泌性高表达过敏毒素C3a的人肾小球足细胞株。结论所建立的过敏毒素C3a分泌性表达慢病毒体系和分泌性高表达过敏毒素C3a的足细胞株,为进一步分析过敏毒素C3a对足细胞的作用创造了条件。Objective To pave the way for investigation of the roles of anaphylatoxin C3a in glomerular podocytes, in the present study, an anaphylatoxin C3a secrectory expression lentiviral system and a podocytes strain highly expressing C3a in a secretory manner was constructed. Methods Anaphylotoxin C3a secretory expression unit was synthesized and cloned into a lentivirus expres- sion vector. After the sequence was confirmed by direct sequencing, recombinant lentivirus was packaged in 293T cells. Infection of HPC, a human glomerular podocytes cell line, with the recombinant lentivirus was carried out. Blasticidin resistant cell clones were screened out. Significantly increased expression and secretion of anaphylatoxin C3a was proved by real-time PCR and enzyme linked immunosorbent assay. Results An anaphylotoxin C3a secrectory expression lentiviral system was correctly established. A podocyte strain highly expressing anaphylotoxin C3a in a secretory manner was obtained. Conclusion An anaphylatoxin C3a secrectory expres- sion lentiviral system and a podocytes strain highly expressing C3a in a secretory manner were successfully constructed. The podocytes strain was very useful for study of the function of C3a/C3aR axis in podocytes. The anaphylatoxin C3a secrectory expression lentiviral system was also useful in study of the function and mechanisms of C3a in other ceils.

关 键 词：过敏毒素 C3A 慢病毒表达体系 分泌性表达 

分 类 号：R363.1[医药卫生—病理学]