慢病毒介导人肝细胞生长因子基因感染BMSCs的实验研究  

RESEARCH OF LENTIVIRAL VECTOR MEDIATED HUMAN HEPATOCYTE GROWTH FACTOR GENE-MODIFIED BONE MARROW MESENCHYMAL STEM CELLS

作　　者：朱金海[1] 陈燕凌[1] 

机构地区：[1]福建医科大学附属协和医院肝胆外科,福州350001

出　　处：《中国修复重建外科杂志》2010年第8期972-976,共5页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：福建省教育厅科研基金资助项目(JA09110)~~

摘　　要：目的构建携带人肝细胞生长因子(human hepatocyte growth factor,hHGF)基因的慢病毒,感染大鼠BMSCs,建立稳定表达hHGF的BMSCs/hHGF细胞。方法从含hHGF基因的质粒pcDNA-hHGF中,用PCR法获取hHGF全长基因,与慢病毒载体pGC-E1分别进行AgeⅠ酶切,产物定向连接,转化后行PCR鉴定和测序,构建hHGF慢病毒表达质粒。脂质体Lipofectamine2000介导慢病毒载体三质粒pGC-E1-hHGF、pHelper1.0、pHelper2.0共转染293T细胞。收集病毒超滤离心浓缩,实时定量PCR测定滴度。将hHGF慢病毒感染BMSCs,荧光显微镜观察荧光表达,确定最佳感染复数(multiplicities of infection,MOI)值,以最佳MOI值感染细胞,流式细胞仪检测感染效率,ELISA法检测细胞培养上清hHGF分泌水平,RT-PCR检测hHGF基因的表达。结果实验成功构建携带hHGF基因的慢病毒,滴度达1×108TU/mL。hHGF慢病毒高效率感染BMSCs,最佳MOI值为10。流式细胞仪检测感染效率达98%,且在感染后28d BMSCs仍稳定表达强烈的绿色荧光。细胞培养上清可检测到hHGF因子,术后5d达高峰,达40.5ng/mL,这种高水平分泌可持续至感染后28d。RT-PCR检测出hHGF基因的表达。结论通过慢病毒载体将hHGF基因稳定感染至BMSCs细胞,可获得长期稳定的表达,并持续分泌hHGF因子。Objective To construct lentiviral vector carrying the human hepatocyte growth factor （hHGF） gene, and then to get hHGF gene/modified bone marrow mesenchymal stem cells （BMSCs） by infecting the BMSCs. Methods The hHGF gene was obtained with PCR from pcDNA-hHGF plasmid. The recombination lentiviral vector plasmid hHGF was constructed with Age I digestion and gene recombinant, then was identified with PCR and sequencing. Mediated by Lipofectamine 2000, the three plasmids system of lentiviral vector including pGC-E1-hHGF, pHelper 1.0, and pHelper 2.0 was co-transfected to 293T cells to produce hHGF gene. The supernatant was collected and concentrated by ultracentrifugation and the titer of lentivirus was measured by real-time quantitative PCR. The BMSCs were infected by the constructed lentivirus and the multiplicities of infection （MOI） was identified with fluorescent microscope, the efficiency of infection with flow cytometry （FCM） analysis, the hHGF level with ELISA analysis, and the expression of hHGF gene with RT-PCR. Results Lentiviral vector carrying hHGF gene was constructed successfully. The titer of lentivirus was 1 × 10 8 TU/mL. The infection efficiency of BMSCs by hHGF lentiviral was high and reached 98% by FCM, and the best MOI was 10. A great mount of green fluorescence was observed with the fluorescent microscope at 28 days after infection. Peak concentration of hHGF secreted by BMSCs/hHGF reached 40.5 ng/mL at 5 days. The concentration could maintain a high level until 28 days after infection. RT-PCR showed that BMSCs/hHGF could express hHGF gene. Conclusion By lentiviral vector, hHGF gene was integrated into BMSCs genome, and it can express stably.

关 键 词：BMSCS 人肝细胞生长因子 慢病毒 基因感染 大鼠 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]