腺病毒介导胰高血糖素样肽-1受体在小鼠骨髓间充质干细胞表达的体外研究

Infection of adenovirus containing GLP-1R gene into mouse mesenchymal stem cells in vitro

目的:观察腺病毒介导胰高血糖素样肽-1受体(GLP-1R)转染小鼠骨髓间充质干细胞(bone marrow mesenchymalstem cells,BM-MSCs)的表达情况及Exendin-4/GLP-1R系统对BM-MSCs细胞增殖及凋亡的影响。方法:利用Ad.Max系统构建含GLP-1R基因的腺病毒表达载体Ad.GLP-1R,感染小鼠BM-MSCs。采用RT-PCR、Western blot和免疫荧光方法检测GLP-1R的表达情况。用Exendin-4刺激感染Ad.GLP-1R的BM-MSCs,通过细胞计数和流式细胞术检测细胞生长曲线、细胞周期及细胞凋亡的变化。结果:腺病毒对小鼠BM-MSCs有较高的感染效率,病毒感染复数(MOI)为400时,感染率高达89.5%。BM-MSCs不表达GLP-1R,感染Ad.GLP-1R后有效表达GLP-1R基因和蛋白。Ad.GLP-1R感染BM-MSCs后,Exendin-4刺激组细胞生长曲线显著高于未刺激组(P<0.05)。细胞周期检测结果显示,刺激组G0/G1期细胞数显著下降、G2/M期细胞数明显升高,增殖指数(S+G2/M)亦显著增加(P均<0.01)。而细胞凋亡率无明显变化。结论:Ad.GLP-1R可以有效感染BM-MSCs,获得高表达GLP-1R的BM-MSCs。Exendin-4可以促进Ad.GLP-1R感染的BM-MSCs增殖。

Objective：To investigate the expression of glucagon like peptide 1 receptor（GLP-1R） in mouse bone marrow mesenchymal stem cells（BM-MSCs） transfected by adenovirus and the effect of Exendin-4/GLP-1R on BM-MSCs proliferation and apoptosis after transfection.Methods： The adenoviral vector containing GLP-1R gene was constructed through the Ad.Max-system and infected the BM-MSCs.The expression of GLP-1R in Ad.GLP-1R-BM-MSCs was examined by RT-PCR,immunofluorescence,and Western blot,respectively.The effects of Exendin-4/GLP-1R on cell growth,cell cycle and apoptosis in Ad.GLP-1R-BM-MSCs were evaluated by cell counting and flow cytometry.Results： BM-MSCs could be effectively infected by adenovirus.When multiplicity of infection（MOI） was 400,the infection efficiency was 89.5%.Mouse BM-MSCs didn＇t express the GLP-1R gene natively.After infected with Ad.GLP-1R,the BM-MSCs expressed the GLP-1R gene and protein effectively,and the cell growth could be stimulated by Exendin-4.Compared with the unstimulated Ad.GLP-1R-BM-MSCs,G0/G1 phase cells in the Exendin-4 stimulation group decreased significantly,G2/M phase cells and proliferation index（S＋G2/M） increased significantly（P 0.01）.But there was no significant difference in the apoptosis rate of two groups.Conclusion： Ad.GLP-1R can infect mouse BM-MSCs effectively and induce the expression of GLP-1R.Exendin-4 can promote the proliferation of BM-MSCs infected with Ad.GLP-1R.