2型重组腺相关病毒介导增强型绿色荧光蛋白在大鼠骨髓间充质干细胞的表达

目的:观察携带增强型绿色荧光蛋白的2型重组腺相关病毒(rAAV_2-EGFP转导骨髓间充质干细胞(MSCs)的效率、表达时间及对细胞增殖的影响。方法:体外培养出MSCs,经传代5次、鉴定后分为2组,rAAV_2组中按感染复数10~6加入0.1 ml rAAV_2-EGFP;PBS组中加入0.1 ml PBS,培养后再传代5次。用流式细胞仪检测2组P5和P10代细胞EGFP转导率,同时绘制2组P10细胞生长曲线。结果:rAAV_2组P5和P10代MSCs转导率分别为21.71%±1.02%、20.34%±1.13%,差异无统计学意义(P=0.862)。生长曲线显示rAAV_2组细胞生长较PBS组缓慢。结论:rAAV_2-EGFP转导MSCs效率可,表达时间长,对细胞增殖稍有影响,标记后的细胞可用于观察干细胞在体内的存活、迁徙及分化。

Green Fluorescent Protein Expression in Rat Bone Marrow Mesenchymal Stem Cells Mediated by Recombinant Adeno-associated Virus Type 2

Objective:To evaluate transduction rates and expression of enhanced green fluorescent protein (EGFP) in mesenchymal stem cells (MSCs) mediated by recombinant adeno-associated virus 2 (rAAV_2),and to ob- serve impacts of the transduction on cell proliferation.Methods:MSCs were dissociated,subcultured and identified by detecting CD44,CD45,and CD90.After subculture for 5 times,the cells were divided into 2 groups,one mixed with 0.1 ml rAAV_2-EGFP with a titer of 10~6,and another incubated with 0.1 ml PBS as control.After another 5 passages,transduction rates of EGFP in the P5 and P10 MSCs were detected by flow cytometry and cell growth curves were generated.Results:The transduction rates of EGFP in the P5 and P10 MSCs of rAAV_2 group were 21.71%±1.02% and 20.34%±1.13% respectively,but no significant differences was found between them (P= 0.862).The growth of MSCs in rAAV_2 group was slower than that of PBS group.Conclusion:EGFP can be ex- pressed in MSCs stably after effective transduction mediated by rAAV_2.The labeled stem cells are applicable to fur- ther investigate the survival,migration and differentiation after transplantation.