大鼠Cx43基因慢病毒表达载体的构建及转染骨髓间充质干细胞表达的观察  

Construction of the recombinant lentiviral carrying rat connexin 43 gene and its expression in bone marrow mesenchymal stem cell of rats

作　　者：李松南[1,2] 毛晓波[1] 冯义柏[1] 王祥[1] 曾秋棠[1] 毛奕[1] 吉庆伟[1] 彭昱东[1] 郭敏[1] 梁志山[1] 

机构地区：[1]华中科技大学协和医院心内科华中科技大学同济医学院心血管病研究所,武汉430022 [2]北京安贞医院心内科,100000

出　　处：《临床心血管病杂志》2010年第11期854-858,共5页Journal of Clinical Cardiology

基　　金：生物靶向治疗湖北省重点实验室课题(No:2008-78)

摘　　要：目的:构建大鼠缝隙连接蛋白43(Cx43)基因的重组慢病毒表达载体,并转染大鼠骨髓间充质干细胞(BMSC),观察Cx43的表达。方法:采用RT-PCR技术获得大鼠Cx43基因,并将其连接到含有增强型绿色荧光蛋白(EGFP)基因的慢病毒表达载体pGC-FU中,重组获得慢病毒质粒pGC-FU-Cx43,将其与辅助包装质粒(pHelper1.0、pHelper2.0)一起共转染293T细胞,包装生产病毒并测定滴度。所获慢病毒感染大鼠BMSC后,采用荧光显微镜和Western blot方法检测Cx43的表达。结果:酶切证实Cx43基因已定向连入目的载体,测序结果显示,所构建的pGC-FU-Cx43重组慢病毒质粒序列与目标序列完全一致。获得的病毒滴度为2×109TU/ml。经分离、纯化得到BMSC;pGC-FU-Cx43转染BMSC后,可见大量EGFP表达,Cx43的表达量明显增加。结论:成功构建并包装获得较高滴度的重组慢病毒pGC-FU-Cx43。pGC-FU-Cx43可高效转染BMSC,Cx43表达增加,且实现稳定转染。Objective：To construct,package and purify the recombinant lentivirus carrying enhanced green fluorescent protein（EGFP）gene and the rat connexin 43 gene（Cx43）.Transfecting the recombinant lentiviral into amplified rat BMSC to observe the Cx43expression level.Method：The rat Cx43 gene was amplified by RT-PCR and connected to the lentiviral expression vector pGC-FU,which is carrying the EGFP gene,to construct the lentiviral vector plasmid pGC-FU-Cx43.Restriction digestion analysis and DNA sequencing was used to verified the correction.The viral particles were generated by cotransfection of 293Tcells with the pGC-FU-Cx43 and two packaging vector（pHelper1.0,pHelper 2.0）and the virus titer was determined by counting the percentage of GFP positive cells.Rat BMSCs were isolated and purified by density gradient centrifugation and adherent cell culture.Flow cytometry was used to identify the surface markers in P3 cells.After transfection of pGC-FU-Cx43 recombinant lentiviral into P3 cells,the expression of EGFP was observed by fluorescence microscope,the level of Cx43 protein in BMSC was determined by western blot assay.Result：The insert orientation of the Cx43 gene in the lentiviral vector plasmid pGC-FU-Cx43 was verified by restriction digestion analysis.Target Cx43 sequences was confirmed by the bulk sequencing.The final titer obtained was 2×109 TU/ml.Increased EGFP expression was observed,expression level of Cx43protein was significantly increased in pGC-FU-Cx43 transfected BMSC.Conclusion：The pGC-FU-Cx43 recombinant lentivirus carrying EGFP gene and Cx43 gene with high viral titer was constructed and packaged successfully.pGC-FU-Cx43 recombinant lentiviral was transfected into BMSC successfully to increase the expression of target protein.

关 键 词：骨髓间充质干细胞 缝隙连接蛋白43 慢病毒 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]