大鼠血小板衍生生长因子C重组腺病毒载体的构建及在内皮祖细胞中的表达  

Construction of the rat recombinant adenovirus vector containing platelet-derived growth factor-C and its expression in the endothelial progenitor cells derived from rat bone marrow

作　　者：李锋[1] 罗文军[1] 唐博[1] 陈以宽[1] 孙建明[1] 付健[1] 

机构地区：[1]重庆医科大学附属第二医院血管外科,400010

出　　处：《重庆医学》2013年第16期1837-1840,共4页Chongqing medicine

基　　金：重庆市卫生局医学科研基金资助项目(2012-2-053)

摘　　要：目的构建含大鼠血小板衍生生长因子C(PDGF-C)基因的pAD-EGFP-PDGF-C的重组腺病毒表达载体,体外转染骨髓源性内皮祖细胞(EPCs)并观察目的基因蛋白及mRNA表达情况。方法通过逆转录聚合酶链式反应技术(RT-PCR)法从pIRES2-EGFP-PDGF-C真核表达载体中获得PDGF-C目的片段,经BP重组与LR重组插入到pAD/CMV/V5-DEST,构建pAD-PDGF-C-IRES2-EGFP腺病毒载体。酶切及DNA测序鉴定后转染HEK293A包装、纯化,测定病毒滴度后转染大鼠骨髓源性EPCs,应用Real-time qPCR、蛋白免疫印迹法(Western blotting)等方法检测PDGF-C蛋白及mRNA的表达。结果核酸内切酶消化及PCR分析证实PDGF-C基因成功插入pAD/CMV/V5-DEST腺病毒载体,转染后荧光显微镜下观察可见绿色荧光蛋白表达,转染48h后实时定量酵素聚合反应技术(Real-time qPCR)测定EPCs中PDGF-C含量明显提高(P<0.05),Wester blotting检测证实在46×103存在特异性条带。结论成功构建了大鼠PDGF-C基因的腺病毒表达载体pAD-EGFP-PDGF-C,体外转染大鼠骨髓源性EPCs能高效表达目的基因产物,为后续研究PDGF-C基因功能以及进一步开展基因治疗奠定了基础。Objective To construct the recombinant adenovirus vector encoding rat PDGF-C gene and to observe the expression of target gene in endothelial progenitor cells derived from rat bone marrow. Methods The gene encoding rat PDGF-C was obtained through RT-PCR from pIRES2-EGFP-PDGF-C. The gene fragment was cloned into pAD/CMV/V5-DEST gateway vector via BP recombin action and LR recombination. After confirmed by PCR and DNA sequencing, the adenovirus vector carrying PDGF-C gene was linearized with Pac I enzyme and transfected into HEK293A cells to package recombinant adenovirus particles. The polymerase chain reaction（PCR）was used to detect target gene. The titre of AD-PDGF-C was measured with the aid of enhanced green fluores-cent protein（EGFP） expression. EPCs derived from rat bone marrow were obtained by adherent culture. The recombinant adenovi-rus was transfected into EPCs.＇The expression of PDGF-C was detected by Real-time qPCR and Western blotting analysis respec-tively. Results The evidence of endonuclease d!gestion and PCR analysis confirmed that PDGF-C gene was correctly inserted into the pAD/CMV/VS-DEST gateway vector. After transfection,the expression of PDGF-C protein in pAD-PDGF-C was significantly higher than that before transfected EPCs group and that of transfected with AD-GFP. Conclusion The recombinant adenovirus vec-tor containing rat PDGE-C was constructed correctly, which can mediate target gene expression in transfected EPCs effectively. This study provides a research basis for constructing genetic transfection tool in follow-up gene therapy study.

关 键 词：血小板衍生生长因子C pAD CMV V5-DEST腺病毒载体 内皮祖细胞 转染 

分 类 号：R363[医药卫生—病理学]