2型腺相关病毒重组绿色荧光蛋白对培养神经干细胞转染效率的研究

为探讨2型重组腺相关病毒(recombinant adeno-associated virus2,rAAV-2)载体能否有效地转染神经干细胞(neural stem cell,NSC),本研究采用含有增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)报告基因的rAAV-2(rAAV-2/EGFP),以感染复数(multiplicity of infection,MOI)分别为1×104、1×105、1×106转染体外培养大鼠胎鼠神经干细胞,在荧光显微镜下观察EGFP的表达。同时,检测子代细胞的活力、增殖倍数、分化情况,以评估rAAV-2对NSC存活、增殖、分化能力的影响。并利用流式细胞仪检测rAAV-2/EGFP对NSC的转染效率。结果显示,转染10d后各转染组可观察到NSC克隆球发出绿色荧光,起始荧光强度不一,同时荧光强度随时间的延长而逐渐增强。转染21d后荧光强度达高峰并维持在高水平。流式细胞仪检测结果表明:MOI为1×104、1×105、1×106时转染率分别为26.34%、50.97%、56.36%,荧光指数(FI)分别为4.01、12.70、14.08。转染组和未转染组细胞培养7、14、21d时其细胞活力、增殖倍数、分化均无统计学差异。本实验初步证明rAAV-2能有效地转染NSC。

TRANSFECTION EFFICIENCY OF RECOMBINANT ADENO-ASSOCIATED VIRUS 2/EGFP IN NEURAL STEM CELL

To investigate the transfection efficiency of recombinant adeno-associated virus 2(rAAV-2) in neural stem cell (NSC), the NSC of rat embryo was infected with rAAV-2 expressing the enhanced green fluorescent protein(EGFP) gene by different multiphcity of infection(MOI=1×104, 1×105, 1×106). The expression of EGFP was detected by fluorescent microscope. The cytotoxicity of rAAV-2 to NSC was evaluated by cell proliferation, cell vitality and cell differentiation. The transfection efficiency was evaluated by flow cytometer. The results showed that EGFP was initially expressed in the neural stem cell-derived neurosphere of all transfected groups 10 d following infected by rAAV-2/EGFP. The level of the expression increased with the increase of MOI and reached peak on 21 d. Transfection efficiency and fluorescent index (FI) were 26.34%, 4.01 (MOI=1×104); 50.97%, 12.70 (MOI=1×105); 56.36%, 14.08 (MOI=1×106), respectively. During the period of the culture, cell proliferation, cell vitality and cell differentiation in the transfected and untransfected groups had no difference. These results indicate that the rAAV-2 can transfect NSC efficiently, and the rAAV-mediated transfer of the gene can be expressed in the NSC.