绿色荧光蛋白基因腺病毒载体转染人骨髓间充质干细胞的实验研究

Experimental study on transfection of adenovirus vector encoding green fluorescent protein into human bone marrow-derived mesenchymal stem cells

目的研究绿色荧光蛋白基因(Ad-GFP)腺病毒载体在人骨髓间充质干细胞(BMSCs)转染和表达的量效关系及对细胞生物学特性的影响,探讨用该载体构建基因修饰BMSCs的可行性。方法在体外分离培养人BMSCs,流式细胞仪检测细胞免疫表型,293细胞包装制备病毒,以不同滴度的Ad-GFP(1×10~3～1×10^(10)PFU/mL)转染BMSCs,细胞计数法分析转染率,倒置显微镜观察细胞形态学改变,CCK8法检测细胞增殖活性,用血清撤离加入β-巯基乙醇诱导转染Ad-GFP的BMSCs向神经元样细胞定向分化。结果3～6代BMSCs表面标志CD34、CD45呈阴性而CD29、CD44呈阳性;当病毒滴度为1×10~7 PFU/mL时转染率为55%,1×10~9及1×10^(10)PFU/mL滴度时转染率均为85%,但1×10^(10)PFU/mL滴度时出现细胞病理现象;7 d荧光表达最强,28 d仍可见荧光表达。荧光显微镜下可见表达Ad-GFP的BMSCs有三种亚群结构;滴度≥1×10~6 PFU/mL时,BMSCs增殖在早期受到抑制,且呈剂量依赖;转染Ad-GFP的BMSCs经β-巯基乙醇诱导可分化为神经元样细胞,神经元特异性烯醇化酶(NSE)阳性。结论合适滴度的Ad-GFP可以高效转染BMSCs,对细胞的生物学特性影响较小,不影响诱导分化功能,BMSCs可以作为用Ad-GFP载体系统进行基因治疗研究的种子细胞。

Objective To explore the transfection of adenovirus encoding green fluorescent protein （Ad-GFP） vector into human bone marrow-derived mesenchymal stem cells （BMSCs）, the dose-effect relationship between them and the impact on biological properties of BMSCs, and further investigate the feasibility of using Ad-GFP vector to construct gene-modified BMSCs. Methods BMSCs were separated to be cultured in vitro. Flow cytometry was applied to detect the immune phenotype of cells; the adenovirus was obtained by packaging cell lines 293; BMSCs were transfected with Ad-GFP of various titers （1×10^3- 1 ×10^10PFU/mL）; cytometry was used to analyze the transfection efficiency; inverted microscope was applied to observe the morphological changes of cells; cell counting kit 8 （CCK8） was applied to detect the activity of cell proliferation; β-mercaptoethanol was added after serum evacuation to induce directed differentiation of Ad-GFP-transfected BMSCs into neuron-like cells. Results CD34 and CD45, and CD29 and CD44, the surface markers of the third- to sixth- passage BMSCs were respectively negative and positive. When the virus titer was I x l 0v PFU/mL, the transfection rate was 55% and when virus titers were 1×10^9 and 1×10^10 PFU/mL, the transfection rate was 85%. In addition, pathological phenomena occurred to cells at the virus titer of 1×10^10PFU/mL. The fluorescent expression achieved its climax at 7 d and could still be seen at 28 d. BMSCs expressing Ad-GFP had three types of subpopulation structures under a fluorescence microscope; when the virus titer was ≥ 1×10^6 PFU/mL, BMSCs proliferation was suppressed at the early stage, in a dose-dependent manner. BMSCs Wansfection with Ad-GFP could be differentiated into neuron-like cells with the induction of β-mercaptoethanol. Neuron-specific enolase （NSE） was positive. Conclusion Ad-GFP with suitable titers can infect BMSCs effectively, and has little impact on biological properties of cells and no impact on the differentiation-inducing function. BMSCs can serve as seed cells during the application of Ad-GFP vector system to the research ofgene therapy.