慢病毒载体介导GFP标记大鼠骨髓间充质干细胞

Labeling of rat bone marrow mesenchymal stem cells with GFP infection by lentiviral vector

目的探究慢病毒载体介导绿色荧光蛋白(green fluorescent protein,GFP)标记大鼠骨髓间充质干细胞(mes-enchymal stem cells,MSCs)的理想条件,以获得稳定高表达GFP的MSCs亚群。方法慢病毒载体介导GFP以25、50、100、200和400的感染复数(multiplicity of infection,MOI)分别作用12 h和24 h感染MSCs,倒置荧光显微镜及流式细胞术检测各组的感染效率和荧光强度,MTT法评价感染对MSCs增殖的影响,从而确定理想的感染条件。平皿克隆套环法挑选理想条件下感染的MSCs单克隆。结果MOI为100、200和400作用24 h时,感染效率较高,分别为88.94%、99.65%、99.42%;MOI为100和200时,感染对MSCs增殖无影响,MOI为400时,感染的MSCs增殖减慢。挑选MOI为200、作用24 h感染组的单克隆细胞,其1月时感染效率为99.95%,且荧光均一、强度很强。结论MOI为200作用24 h是慢病毒载体介导GFP标记MSCs的理想条件;通过细胞单克隆技术可获得稳定高表达GFP的MSCs亚群。

Objective To determine the optimal condition for labeling rat bone marrow mesenchymal stem cells（MSCs） with green fluorescent protein（GFP）infection by lentiviral vector so as to establish a subgroup of MSCs which have a high level of GFP expression.Methods MSCs were infected with GFP by lentiviral vector for 12h or 24h at different MOI（25,50,100,200 and 400）.The infection efficiency and fluorescence intensity were analyzed by inverted fluorescent microscopy and flow cytometry.The effect of infection at different MOI on proliferation of MSCs was evaluated by MTT.Based on those mentioned above,we could determine the optimal condition for infection.Then single cell clones of MSCs labeled with GFP under optimal condition were selected by using cloning rings.Results The efficiency of infection for 24h at MOI 100,200 and 400 was 88.94%,99.65% and 99.42%,respectively.The infection had no significant effect on the proliferation of MSCs infected at MOI 100 or 200,compared with MSCs without infection.However,those MSCs infected at MOI 400 proliferated slowly.The rate of GFP expression on single-cell clone of MSCs infected for 24h at MOI 200 was 99.95%,and their fluorescence intensity was strong and uniform.Conclusion Infection for 24h at MOI 200 is optimal for labeling rat bone marrow MSCs with GFP by a lentiviral vector.A subgroup of MSCs which have a stably high level of GFP expression can be obtained by single cell cloning technique.