慢病毒介导血管内皮细胞生长因子165/骨形态发生蛋白2双基因转染犬骨髓间充质干细胞的研究

摘要

目的 探讨慢病毒介导血管内皮细胞生长因子165(VEGF165)/骨形态发生蛋白2(BMP-2)双基因对犬骨髓间充质干细胞(BMSCs)的转染及其表达情况. 方法 分离培养犬BMSCs,构建携带VEGF165和BMP-2基因的重组慢病毒表达载体,利用293T细胞包装、生产携带VEGF165和BMP-2基因的重组慢病毒(Lv-BMP、Lv-VEGF),分为未转染组、VEGF165组、BMP-2组及VEGF165+ BMP-2组(联合转染组),后三组分别转染第三代犬BMSCs.于转染后7d运用实时荧光定量RT-PCR检测各组BMSCs中VEGF165、BMP-2 mRNA表达;于转染后5,28 d运用Western blotting检测BMSCs中VEGF165、BMP-2蛋白表达情况;运用细胞四唑盐比色法(MTT)测定转染后各组BMSCs生长情况并绘制生长曲线. 结果 RT-PCR检测结果显示,VEGF165 mRNA仅在VEGF165组与双基因联合转染组中存在高表达,BMP-2 mRNA仅在BMP-2组与双基因联合转染组中存在高表达,且各两组间mRNA的表达差异无统计学意义(P＞0.05);Western blotting法结果均显示,VEGF165蛋白仅在VEGF165组与双基因联合转染组中有特异性表现,BMP-2蛋白仅在BMP-2组与双基因联合转染组中有特异性表现;MTT测定显示各组细胞在培养初期有2～3d左右的相对静止期,从第3天起各组细胞均增殖迅速,呈现对数生长,7d后进入平台期,细胞增殖减缓,吸光度(A)值比较发现转染与未转染细胞间生长情况差异无统计学意义(P＞0.05). 结论 慢病毒介导的VEGF165/BMP-2双基因修饰方法可成功转染BMSCs,并得到持续高表达VEGF165/BMP-2的BMSCs.%Objective To transfect the dog bone mesenchymal stem cells (BMSCs) with lentivirus-mediated vascular endothelial growth factor 165 (VEGF165) and bone morphogenetic protein 2 (BMP-2) and observe the expressions of VEGF165 and BMP-2 in BMSCs.Methods Lentivirus vectors carrying VEGF165 and BMP-2 genes were constructed and used to transfect dog third-generation BMSCs,including untransfected group,VEGF165 group,BMP-2 group and double-gene (VEGF165 + BMP-2) group.Expressions of VEGF165 and BMP-2 mRNA were detected by RT-PCR 7 d after the transfection.Expressions of VEGF165 and BMP-2 protein were detected by Western blotting 5 d and 28 d after the transfection.Cell growth was detected by MTT assay.Results RT-PCR showed high expression of VEGF165 mRNA in VEGF165 and double-gene groups,and BMP-2 mRNA in BMP-2 and double-gene groups,but there was no significant difference in group comparison (P ＞ 0.05).Western blotting showed specific performance of VEGF165 protein in VEGF165 and double-gene groups,and BMP-2 protein in BMP-2 and double-gene groups.In MTT assay cells were found to have 2-3 d relatively stationary phase in the early period,proliferated rapidly at 3 d,reaching the logarithmic growth,and entered the plateau phase with slow propagation.There was no significant difference in absorbance value before and after the transfection (P ＞ 0.05).Conclusion BMSCs with highly expressed VEGF165 and BMP-2 can be successfully obtained by lentiviral transduction.