绿色荧光蛋白基因腺病毒载体转染大鼠骨髓间充质干细胞的实验研究

Transfection of rat bone marrow mesenchymal stem cell with green fiuorescent protein by adenovirus vector

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（1×10^3～1×10^10PFU／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、CIM5阴性而CD29、CD44阳性，当病毒滴度为1×10^7PFU／ml时转染率为55％，为1×10^9及1×10^10PFU／ml转染率均为85％，但1×10^10PFU／ml时出现细胞病理现象，7d荧光表达最强，28d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 - relationship between transfection and expression rat bone marrow stem cell with Ad - GFP and the change of cell biology property and to study the feasibility of constructing gene - modified BMSCs using Ad - GFP vector. Methods BMSCs were isolated and cuhured in vitro and cell immunophenotypes were determined by flow cytometry. Adenovirus was obtained by packaging 293 cell. BMSCs was transfected at various titers （ 1 × 10^-3～ 1 × 10^10 PFU/ml）. Transfection efficiency was analyzed by flow cytometry and cell proliferation was detected by CCK - 8 kits. BMSCs transfected with Ad - GFP were induced to differentiate into neuron type cells withdraw of serum and addition of β - mereaptoethanol. Results The surface markers of 3 ～ 6 generation BMSCs, CD34 and CIM5, were negative. However CD29 and CD44 were positive. When the virus titers were 1 × 10^7PFU/ml, the infection rate was 55%. When the titers were 1 × 10^9 and 1 × 10^10PFU/ml, the infection rate was 85%. However,the latter titer was associated with cell pathology. The strongest expression of fluorescence occurred at day 7 and could still be seen at day 28. The BMSCs infected with Ad - GFP differentiated into neuron type ceU and the neuron - specific enolase was positive after addition of β - mercaptoethanol. Conclusion Suitable titers of Ad - GFP can infect BMSC effectively, have little influence on the biology property and do not hamper differentiation function of the cell. BMSCs can serve as seeds cell for gene therapy when utilizing Ad- GFP vector system.