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目的研究腺病毒载体绿色荧光蛋白基因(Ad-GFP)在大鼠骨髓间充质干细胞(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大鼠骨髓间充质干细胞,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(1×103~1×1010PFU/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,CCK-8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向神经样细胞定向分化。结果3~6代BMSCs表面标志CD34、CD45阴性而CD29、CD44阳性,当病毒滴度为1×107PFU/ml时转染率为55%,为1×109及1×1010PFU/ml转染率均为85%,但1×1010PFU/ml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,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Ad-GFP载体系统进行基因治疗的种子细胞。
Objective To study the dose-response relationship of Ad-GFP transfection and expression in rat bone marrow mesenchymal stem cells (BMSCs) and its effect on the biological characteristics of the cells, and to explore the feasibility of using this vector to construct genetically modified BMSCs Feasibility. Methods Rat bone marrow mesenchymal stem cells (MSCs) were isolated and cultured in vitro. Cell phenotypes were detected by flow cytometry. 293 cells were packaged for virus production and transfected with Ad-GFP (1 × 103 ~ 1 × 1010 PFU / ml) BMSCs were transfected with BMSCs. Transfection efficiency was analyzed by cell counting method. Morphological changes were observed by inverted microscope. Cell proliferation was detected by CCK-8 assay. BMSCs transfected with Ad-GFP were induced to differentiate into neuron-like cells by serum evacuation and β-mercaptoethanol. Results On the 3rd to 6th generation, the surface markers of BMSCs were negative for CD34, CD45 and positive for CD29 and CD44. The transfection rate was 55% when the virus titer was 1 × 107 PFU / ml, and the transfection rates were 1 × 109 and 1 × 1010 PFU / ml respectively Was 85%. However, 1 × 1010PFU / ml showed cytopathology, the fluorescence intensity was the highest on the 7th day and the fluorescence was still visible on the 28th day. BMSCs transfected with Ad-GFP were differentiated into neuron-like cells induced by β-mercaptoethanol , Neuron specific enolase (NSE) positive. Conclusion Ad-GFP with the proper titer can efficiently transfect BMSCs with little effect on the biological characteristics of the cells. It does not affect the differentiation-inducing function and BMSCs can be used as seed cells for gene therapy by Ad-GFP vector system.