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

摘要] 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(BMSCc)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BMSCc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(1×103-1×1010 PFU/ml)转染BMSCc，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- 巯基乙醇诱导感染Ad-GFP的BMSCc向神经样细胞定向分化。结果：3-6代BMSCc表面标志CD34、CD45阴性而CD29、CD44阳性，当病毒滴度为1×107PFU/ml时感染率为55%，为1×109及1×1010PFU/ml感染率均为85%，但1×1010PFU/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，感染Ad-GFP的BMSCc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可以高效感染BMSCc，对细胞的生物学特性影响较小不影响诱导分化功能，BMSCc可以作为Ad-GFP载体系统进行基因治疗的种子细胞。

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

Abstract] Objective:To explore the dose-relationship between transfection and expression rat bone marrow stem cell with ad –GFP and the change of cell biology property ,and to explore the feasibility of useing ad-GFP vector construc gene modify BMSCc.Methods:separate and culture BMSCc in vitro,detect the cell immunophenotype by flow cytometry ,the adenvirus was obtained by packaging 293 cell, BMSCc was transfected by various titers(1×103-1×1010 PFU/ml) , Cytometry analysis the transfection efficiency ,detection the cell proliferation by CCK8 kits, induced BMSCc which infected ad-GFP differentiate into neuron type cell by evacuated serum and addedβ-mercaptoethanol .Results:the surface marker of 3-6 generation BMSCc, CD34、CD45 were negative,however CD29、CD44 were positive,when the virus titers were 1×107PFU/ml,the infection rate were 55%,were 1×109and 1×1010PFU/ml,the infection rate were 85%,but the latter occurred cell pathology phenomenon, the most strong expression of fluorescent in the 7days , and the fluorescent can still be seen in 28days,the BMSCc which infected ad-GFP can differentiate into neuron type cell and the neuron - specific enolase was positive after added β-mercaptoethanol.Conclusion:suitable titers of Ad-GFP can infect BMSCc effectively ,have little influence on the biology property and did not hamper the induced differentiation function of the cell, BMSCc can serve as seeds cell when utilize ad –GFP vector system undertake gene therapy.