重组腺病毒介导肝细胞生长因子转染大鼠骨髓间充质干细胞的实验研究

Recombinant adenovirus-mediated hepatocyte growth factor transfection of rat bone marrow mesenchymal stem cells:an experimental study

目的评价重组腺病毒介导肝细胞生长因子(Ad-HGF)对大鼠骨髓间充质干细胞(BMSC)的转染效率及对干细胞增殖的影响。方法1只雄性Wistar大鼠,鼠龄4周,体质量约90 g。采用密度梯度离心法结合贴壁法分离培养大鼠BMSC,流式细胞仪进行鉴定;携带绿色荧光蛋白基因的重组腺病毒(Ad-GFP)转染BM-SC,探索最佳感染指数(MOI),激光共聚焦显微镜观察转染效果,四甲基偶氮唑盐微量酶反应比浊法(MTT)测定转染对BMSC增殖的影响;在最佳MOI时Ad-HGF转染BMSC,酶联免疫吸附试验(ELISA)法测定人HGF在不同时间的表达。结果成功培养大鼠BMSC,GFP成功表达,不影响细胞增殖(P>0.05);转染效率随MOI增大逐渐增加,在MOI=200时,效率达88.42%;Ad-HGF成功高效率转染MSC,HGF基因得到持续高表达,48 h时达最高水平。结论Ad-HGF成功高效率转染大鼠BMSC,外源基因得到持续表达,不影响干细胞活性。

Objective To evaluated the transfection efficiency of recombinant adenovirus-mediated hepatocyte growth factor(Ad-HGF)on rat bone marrow mesenchymal stem cells(BMSC)and its effect on stem cell proliferation.Methods A male Wistar rat,aged 4 weeks old,weighing about 90 g was included in the study.Density gradient centrifugation combined with adherence method was used to separate and culture rat BMSCs,and flow cytometry was used for identification.Ad-GFP was used to transfect BMSCs,and the optimal multiplicity of infection(MOI)was determined.Laser confocal microscope was used to examine the transfection effect,and the MTT method was used to determine the effect of transfection on the proliferation of BMSCs.At the optimal MOI,Ad-HGF was transfected into BMSC,and the expression of human HGF at different time points was determined by ELISA.Results The rat BMSCs were successfully cultured and GFP was successfully expressed without affecting cell proliferation(P>0.05).The transfection efficiency gradually increased along with the increasing MOI.When MOI=200,the efficiency reached 88.42%.Ad-HGF was successfully transfected into MSC with high efficiency,and the HGF genes were persistently and highly expressed,reaching the highest level at 48 h.Conclusion Ad-HGF may successfully transfect into rat BMSCs with high efficiency,and exogenous genes are persistently expressed without affecting stem cell activity.