碱性成纤维细胞生长因子-慢病毒真核表达载体的构建及转染  

Construction and transfection of lentiviral eukaryotic expression vector carrying basic fibroblast growth factor

作　　者：胡杨[1] 盛磊[2] 马莹[1] 何惠宇[1] 阿布力孜.阿布杜拉 阿尔孜古丽[1] 

机构地区：[1]新疆医科大学第一附属医院,新疆维吾尔自治区乌鲁木齐市830054 [2]新疆医科大学地方病重点分子生物学实验室,新疆维吾尔自治区乌鲁木齐市830054

出　　处：《中国组织工程研究与临床康复》2011年第6期1009-1014,共6页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：新疆维吾尔自治区科技攻关项目资助(200533118);项目名称:组织工程骨用于即刻牙种植骨结合的实验研究;新疆维吾尔自治区高校科研计划科学研究重点项目(xjEDU2009I22);课题名称:bFGF基因转染的骨髓间充质干细胞复合异种煅烧骨修复颌骨缺损的实验研究;乌鲁木齐市科学技术计划项目(Y09131002);课题名称:牙周植骨术联合固定义齿夹板保留牙周炎松动牙的临床应用研究~~

摘　　要：背景:以往研究多采用质粒载体,由于其转染效率不高,且转染时需借助脂质体转染剂进行转染,转染具有细胞毒性,操作复杂等难以应用于临床。目的:构建携带人碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)基因的慢病毒载体,转染成骨方向诱导的骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs),鉴定bFGF基因表达。方法:实验组:设计人bFGF基因引物,用Trizol法提取胎盘组织RNA,用RT-PCR的方法扩增出bFGF基因,连接至pLenti6/V5-D-TOPO表达质粒,经Xho-Ⅰ、BamH-Ⅰ双酶切和DNA测序证实质粒正确构建。在脂质体转染剂Lipofectamine2000的介导下,将bFGF-pLenti6/V5-D-TOPO表达质粒同包装质粒pLP1、pLP2、包膜质粒pLP/VSVG共转染293FT细胞株,收集bFGF-慢病毒上清转染诱导后第2代的兔BMSCs。对照组:设计GFP基因引物,以GFP-PMSLV-Plazmid为模板,PCR的方法扩增出GFP基因,连接至pLenti6/V5-D-TOPO表达质粒,构建GFP-慢病毒载体,并转染BMSCs。RT-PCR和Wetern-blot方法检测bFGF、GFP基因的表达。结果与结论:转染48h后,对照组BMSCs可见绿色荧光蛋白表达;实验组BMSCs在转染15d后,RT-PCR方法扩增出bFGF基因,Western-blot检测出目的蛋白表达。提示成功构建携带人bFGF和GFP基因的真核表达载体,建立转染兔BMSCs的方法。BACKGROUND：Previous studies mainly used plasmid vector.Due to its low transfection efficiency,liposome transfection reagent is needed.The transfection has cytotoxicity,with complex operation.OBJECTIVE：To construct lentiviral vector carrying human basic fibroblast growth factor（bFGF） gene,to transfect ossification-induced bone marrow mesenchymal stem cells（BMSCs） and to identify the expression of bFGF gene.METHODS：Experimental group：bFGF gene primers were designed,total RNA was extracted from placental tissue using TRIzol.The bFGF gene amplified by RT-PCR,and the PCR product was connected to the pLenti6/V5-D-TOPO  expression plasmid,which proved correctly constructed by the Xho-Ⅰand BamH-Ⅰ double digestion and DNA sequencing.With the promotion of Lipofectamine 2000,bFGF-pLenti6/V5 plasmid and packaging plasmid pLP1,pLP2,pLP/VSVG cotransfected 293FT cell line,bFGF-lentivirus supernatant was collected and infected the passageⅡ BMSCs.Control group：the green fluorescent protein（GFP） gene was amplified by PCR from GFP-PMSLV-Plazmid,GFP gene was connected to pLenti6/V5-D-TOPO  expression plasmid,and transfected into the BMSCs.The bFGF and GFP gene expression was detected by RT-PCR and Western-blot assay.RESULTS AND CONCLUSION：At 48 hours after transfection,GFP of the control BMSCs was visible.At 15 days after transfection,the bFGF expression of the experimental BMSCs was detected by RT-PCR and Western-blot.These indicated that lentiviral vectors carrying human bFGF and GFP were successfully constructed,and a method of transfecting rabbit BMSCs was constructed.

关 键 词：碱性成纤维细胞生长因子 骨髓间充质干细胞 基因 慢病毒 转染 

分 类 号：R394.2[医药卫生—医学遗传学]