C3a分泌性表达载体及过表达C3a肾小管上皮细胞株的构建  

Construction of C3a secrectory expression vector and tubular epithelial cell line stably expressing C3a

作　　者：郑敬民[1] 尹广[1] 恽时锋[2] 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,南京210002 [2]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比较医学科,南京210002

出　　处：《医学研究生学报》2015年第6期584-589,共6页Journal of Medical Postgraduates

基　　金：国家自然科学基金(81370828)

摘　　要：目的已有的研究表明包括糖尿病肾病在内的多种肾病患者肾小管上皮细胞存在C3a/C3aR轴过度活化现象,但有关C3a/C3aR轴过度活化在肾小管上皮细胞中的病理意义未知。文中构建分泌性过表达C3a的肾小肾小管上皮细胞株,为探讨各种病理情况下C3a/C3aR轴过度活化的病理意义提供细胞模型。方法设计合成人C3a分泌性表达单元,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C3a分泌性表达载体p Lenti6.3-C3a-IRES2-EGFP;将p Lenti6.3-C3a-IRES2-EGFP和包装质粒共转染293 T细胞,包装成C3a表达重组慢病毒LV-C3a;以LV-C3a感染人肾小管上皮细胞系HK2,根据LV-C3a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ELISA方法对稳定转染细胞克隆的C3a表达水平和分泌情况进行分析,从中鉴定出稳定分泌性过表达C3a的人肾小管上皮细胞株。结果 1成功构建了序列完全正确的C3a分泌性表达载体p Lenti6.3-C3a-IRES2-EGFP;2成功进行了重组慢病毒包装,得到了高滴度(5×108个/m L)的C3a表达重组慢病毒LV-C3a;3成功进行了LV-C3a对HK2细胞的转染,得到了稳定转染LV-C3a的HK2细胞株;基于荧光定量PCR和ELISA的分析证实,与HK2细胞比较,HK2-C3a细胞株中C3a mRNA表达水平显著升高[(1.0±0.5)vs(1 321.0±18.0),P<0.01],分泌的C3a水平显著升高[(0.3±0.2)ng/m L vs(249.0±37.0)ng/m L,P<0.01]。结论成功构建的C3a分泌性慢病毒表达载体和分泌性过表达C3a的人肾小管上皮细胞株为进一步研究各种病理情景中C3a/C3aR过度活化在人肾小管上皮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C3a/C3aR过度活化在其他细胞中的病理意义创造了条件。Objective Evidence from previous studies indicated that over-activation of C3a/C3 aR axis existed in the renal tubular epithelial cells of patients with renal diseases including diabetic nephropathy. However,the pathological significance of over-activating C3 a / C3 aR axis still remains to be elucidated. In this study,we constructed a renal tubular epithelial cell line over-expressing C3 a in a secretory manner in order to provide a cell model to investigate the pathological significance of over-activating C3 a / C3 aR axis under various pathological scenes. Methods We designed a synthesized C3 a secretory expression unit and cloned it into the multiclonal site of lentivirus expression vector p Lenti6. 3-MCS-IRES2-EGFP. After identification by sequencing,recombinant lentivirus was packaged by using p Lenti6. 3-C3a-IRES2-EGFP and packaging plasmid in 293 T cells. Then,the recombinant lentivirus was used to infect HK2,a cell line of human renal tubular epithelial cells. After screening in medium with blasticidin,blasticidin resistant cell clones were obtained. Real-time PCR and ELISA method were applied to analyze the expression and secretion of stable transfected cells cloned C3 a and identify renal tubular epithelial cell lines with stable over-activating C3 a. Results 1C3 a secretory expression unit was successfully synthesized and correctly cloned into the multi-clonal site of p Lenti6. 3-IRES2-EGFP; 2C3 a secrectory expression recombinant lentivirus LV-C3 a was successfully packaged with a high titer of 5 × 108/ m L; 3HK2 Cell clones resistant for blasticidin were obtained; according to the analysis of Real-time PCR and ELISA,the C3 a mRNA level in HK2-C3 a cell lines was significantly higher than that of HK2 cells（ 1. 0 ± 0. 5 vs 1321. 0 ± 18. 0,P〈0. 01） and the secreted C3 a level increased significantly（ [0. 3 ± 0. 2]ng /m L vs [249. 0 ± 37. 0]ng / m L,P〈0. 01）. Conclusion The present study successfully constructed C3 a secretory expression vector p Lenti6. 3-C3a-IRES2-EGFP and C3 a over-e

关 键 词：C3A C3aR 慢病毒 肾小管上皮细胞 过表达 

分 类 号：R692[医药卫生—泌尿科学]