TGF-β1、EGFP双基因修饰骨髓间充质干细胞的体外实验研究  

Expression of Eukaryotic Vector Carrying TGF-β1 and EGFP Genes in BMSCs.

作　　者：曹颖光[1] 王戎[1] 宋珂[1] 王华均[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院口腔系,湖北武汉430030

出　　处：《口腔医学研究》2007年第1期28-31,共4页Journal of Oral Science Research

基　　金：武汉市科技攻关计划项目(编号:20036004)

摘　　要：目的:构建pTGF-β1-IRES2-EGFP双顺反子真核表达载体,检测其在骨髓间充质干细胞(BMSCs)中的表达,为研究基因修饰细胞回植后的迁移转归及成骨作用提供实验基础。方法:构建p TGF-β1-IRES2-EGFP双顺反子真核表达载体,p TGF-β1-IRES2-EGFP与pIRES2-EGFP分别转染BMSCs,并设空白对照。荧光显微镜和细胞免疫组化分别检测GFP和TGF-β1的表达。结果:成功地构建含TGF-β1基因和EGFP基因的真核表达载体。G418筛选10-15 d后有抗性克隆形成。pTGF-β1-IRES2-EGFP转染组抗性克隆中,GFP表达的同时有TGF-β1的表达,而部分抗性克隆(约20%)中有TGF-β1表达的却无GFP表达。pIRES2-EGFP转染组的抗性克隆中有EGFP表达,但无TGF-β1表达。结论:利用IRES构建的含TGF-β1基因和GFP基因的双顺反子真核表达载体,能在部分BMSCs中同时获得表达,可以作为TGF-β1基因修饰BMSCs体内回植后追踪其成骨作用的方法。Objective： To construct a bicistronic eukaryotic co - expression plasmid carrying TGF -β1 and enhanced green fluorescent protein （EGFP） and observe its expression in bone mesenchymal stem cells（BMSCs） in vitro. Methods： TGF -β1 expression unit was inserted into pIRES2 - EGFP to produce the bicistronic eukaryotic co - expression plasmid. The recombinant plasmid and pIRES2 - EGFP were transfected into BMSCs, respectively, which were detected by fluorescence microscope and cell immunohistochemical staining. The untransfected BMSCs served as control group. Results： The eukaryotic vector carrying TGF -β1 and EGFP genes was successfully constructed. Positive clones were obtained by G418 screening after 10 - 15 days. The clones group which was transfected by pTGF -β1 - IRES2 - EGFP expressed EGFP and TGF-β1 simultaneiously. However, some clones （20%） only expressed TGF-β1 but no EGFP. Another group which was transfected by pIRES2 - EGFP expressed EGFP but no TGF - 61. Conclusion ： The bicistronic eukaryoutic vector containing TGF - 61 and EGFP could express TGF - 61 and EGFP in some of BMSCs,which provided a method to study the migration and the osteogenesis of TGF - 61 gene - modified BMSCs in vivo.

关 键 词：转化生长因子Β1 增强型绿色荧光蛋白 骨髓间充质干细胞 

分 类 号：R318[医药卫生—生物医学工程]