重组人肝细胞生长因子在人脐带间质干细胞中高效表达  

High level expression of recombinated hepatocyte growth factor in human umbilical cord mesenchymal stem cells

作　　者：刘安民[1] 罗铭[2] 李国[3] 蔡望青[1] 李方成[1] 邓跃飞[1] 潘伟生[3] 

机构地区：[1]中山大学孙逸仙纪念医院神经外科,广东广州510120 [2]中山大学孙逸仙纪念医院肿瘤科,广东广州510120 [3]香港中文大学

出　　处：《中国病理生理杂志》2008年第12期2450-2454,共5页Chinese Journal of Pathophysiology

基　　金：香港中文大学健康科学研究院干细胞研究基金资助项目

摘　　要：目的:构建能表达人肝细胞生长因子(hHGF)的pWPI-hHGF载体,并将含hHGF基因的重组慢病毒载体转染人脐带间质干细胞(hUCMSCs),观察人脐带间质干细胞中hHGF的表达情况。方法:将携带目的基因hHGF的pUC-SRα/hHGF质粒亚克隆到真核细胞表达载体pWPI载体上,构建重组质粒pWPI-hHGF。基因测序进行HGF的鉴定。用磷酸钙沉淀法,将pWPI-hHGF、pAX2、pMD2G共同转染包装细胞293T细胞,获得携带目的基因hHGF和GFP基因的重组慢病毒pWPI-hHGF。将pWPI-hHGF及阳性对照质粒pWPI-GFP分别用Lipo-fectamin 2000介导转染体外培养的hUCMSCs。在荧光显微镜下计数,确定阳性对照质粒的转染数,从而估计该基因的转染效率。蛋白印迹法检测HGF和GFP蛋白的表达,ELISA法检测细胞上清液hHGF含量。结果:DNA测序显示hHGF基因成功地插入到pWPI载体中。包装细胞293T转导pWPI-HGF质粒后,转染阳性率达100%。阳性对照质粒转染人脐带间质干细胞24 h后,在荧光显微镜下观察,其转染效率达80%以上。蛋白印迹法检测靶细胞中hHGF表达呈强阳性,而对照组表达量极低,两者差异显著(P<0.01)。检测收集转染hHGF后的人间质干细胞上清液中hHGF的表达含量明显高于对照组(P<0.01)。结论:构建的在真核细胞内表达hHGF的重组质粒pWPI-hHGF转染人脐带间质干细胞,能获得hHGF基因在人脐带间质干细胞中大量、稳定的表达。AIM： To construct a lentiviral vector encoding human hepatocyte growth factor （hHGF） for transfection, and to observe the expression of hHGF in human umbilical cord mesenchymal stem cells. METHODS： pUC - SRα/hHGF was subcloned into the expression vector pWPI to construct recombinant pWPI - hHGF. hHGF was identified by gene sequence. Recombinant lentivirus was produced by pWPI - hHGF, pAX2 and pMD2G altogether transient transfection into 293T cells using calcium phosphate method. The pWPI - hHGF and the contructed pWPI - GFP were transfected into human umbilical cord mesenchymal stem cells by the Lipofectamin 2000. Through counting by the fluorescent microscope, the efficiency of the transfection was identified. The expressions of hHGF and GFP in human umbilical cord mesenchymal stem cells were also detected. The concentration of hHGF in cell culture medium was determined by enzyme linked immunosorbent assay （ELISA）. RESULTS： DNA sequence showed that hHGF cDNA was correctly inserted into pWPI vector. The positive rate of hHGF transfecting 293T cells was 100 %. Bright green fluorescence in the transfected cells was observed under the fluorescent microscope after 24 h transfection with lentiviral plasmid pWPI - hHGF - GFP, and the transfection rate reached 80%. The difference was distinct between the pWPI - hHGF group and control group in the secretive level of hHGF by Western blotting and the ELISA （ P 〈 0. 01 ）. CONCLUSION ： The recombinant pWPI - hHGF plasmid was successfully constructed and efficient, stable and ectopic expression of hHGF was accomplished in human umbilical cord mesenchymal stem cells.

关 键 词：肝细胞生长因子 慢病毒载体 载体构建 脐带间质干细胞 基因表达 

分 类 号：R363[医药卫生—病理学]