东南国防医药
東南國防醫藥
동남국방의약
JOURNAL OF SOUTHEAST CHINA NATIONAL DEFENCE MEDICAL SCIENCE
2014年
6期
561-565
,共5页
郑敬民%尹广%赵文紧%刘志红
鄭敬民%尹廣%趙文緊%劉誌紅
정경민%윤엄%조문긴%류지홍
C3 a受体%慢病毒%肾小球足细胞%过表达
C3 a受體%慢病毒%腎小毬足細胞%過錶達
C3 a수체%만병독%신소구족세포%과표체
C3aR%lentivirus%podocytes%over-expression
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pLenti6．3-MCS-IRES2-EGFP的多克隆位点，构建成C3aR表达载体pLenti6．3-C3aR-IRES2-EGFP；将pLenti6．3-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pLenti6．3-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C3 aR表达载体和过表达C3 aR的人肾小球足细胞株，为进一步研究C3 aR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C3 aR在其他细胞中的生理、病理意义创造了条件。
目的：構建過錶達C3a受體（C3aR）的腎小毬足細胞株，為探討C3aR在腎小毬足細胞中的確切生理和病理意義提供細胞模型。方法設計閤成人C3aR 錶達單元，將其剋隆到慢病毒錶達載體pLenti6．3-MCS-IRES2-EGFP的多剋隆位點，構建成C3aR錶達載體pLenti6．3-C3aR-IRES2-EGFP；將pLenti6．3-C3aR-IRES2-EGFP和包裝質粒共轉染293 T細胞，包裝成C3aR錶達重組慢病毒LV-C3aR；以LV-C3aR感染人腎小毬足細胞繫HPC，根據LV-C3aR上帶有殺稻瘟菌素抗性基因的特點，以殺稻瘟菌素篩選穩定轉染細胞剋隆；利用熒光定量PCR和細胞免疫化學分析方法對穩定轉染細胞剋隆的C3aR錶達水平進行分析，從中鑒定齣穩定過錶達C3aR的人腎小毬足細胞株。結果成功構建瞭C3aR錶達載體pLenti6．3-C3aR-IRES2-EGFP；得到瞭高滴度的C3aR錶達重組慢病毒LV-C3aR；成功構建瞭過錶達C3aR的人腎小毬足細胞株HPC-C3aR。結論成功構建C3 aR錶達載體和過錶達C3 aR的人腎小毬足細胞株，為進一步研究C3 aR過錶達在人腎小毬足細胞中的病理意義提供瞭很好的細胞模型，也為進一步開展C3 aR在其他細胞中的生理、病理意義創造瞭條件。
목적：구건과표체C3a수체（C3aR）적신소구족세포주，위탐토C3aR재신소구족세포중적학절생리화병리의의제공세포모형。방법설계합성인C3aR 표체단원，장기극륭도만병독표체재체pLenti6．3-MCS-IRES2-EGFP적다극륭위점，구건성C3aR표체재체pLenti6．3-C3aR-IRES2-EGFP；장pLenti6．3-C3aR-IRES2-EGFP화포장질립공전염293 T세포，포장성C3aR표체중조만병독LV-C3aR；이LV-C3aR감염인신소구족세포계HPC，근거LV-C3aR상대유살도온균소항성기인적특점，이살도온균소사선은정전염세포극륭；이용형광정량PCR화세포면역화학분석방법대은정전염세포극륭적C3aR표체수평진행분석，종중감정출은정과표체C3aR적인신소구족세포주。결과성공구건료C3aR표체재체pLenti6．3-C3aR-IRES2-EGFP；득도료고적도적C3aR표체중조만병독LV-C3aR；성공구건료과표체C3aR적인신소구족세포주HPC-C3aR。결론성공구건C3 aR표체재체화과표체C3 aR적인신소구족세포주，위진일보연구C3 aR과표체재인신소구족세포중적병리의의제공료흔호적세포모형，야위진일보개전C3 aR재기타세포중적생리、병리의의창조료조건。
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 .
