中国组织工程研究与临床康复2011，Vol.15Issue(6)：1009-1014,6.DOI:10.3969/j.issn.1673-8225.2011.06.014
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
摘要
Abstract
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 TRIzoI.The bFGF gene amplified by RT-PCR, and the PCR product was connected to the pLenti6N5-D-TOPO (R) expression plasmid,which proved correctly constructed by the Xho- Ⅰ and 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 passage Ⅱ 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 (R) expression plasmid, and transfected into the BMSCs. The bFGF and GFP gene expression was detected by RT-PCR and Western-blot assay.RESU LTS 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
医药卫生引用本文复制引用
胡杨,盛磊,马莹,何惠宇,阿布力孜·阿布杜拉,阿尔孜古丽..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[J].中国组织工程研究与临床康复,2011,15(6):1009-1014,6.基金项目
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(xj EDU2009I22),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 （200533118）
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. （Y09131002）
