腺相关病毒介导增强型绿色荧光蛋白基因体外转染兔结膜上皮细胞

目的:研究2型重组腺相关病毒( recombinant adeno-associated virus 2,rAAV2)载体介导增强型绿色荧光蛋白基因(enhanced green fluorescent protein,EGFP) 对体外培养兔结膜上皮细胞的转染和表达情况,为后续研究提供依据。方法:体外培养兔结膜上皮细胞,rAAV2-EGFP 按感染复数(multiplicity of infection ,MOI)为104、105、106 转染第2代兔结膜上皮细胞,转染后第1 、3 、5 、7日倒置荧光显微镜下观察细胞中EGFP表达情况,计算转染率。MTT方法检测rAAV2-EGFP转染对细胞增殖的影响。结果:随着MOI值增大及转染时间延长,EGFP 表达效率逐渐增高,转染后第7～8日达到高峰并维持。MTT检测各MOI组与对照组差别无统计学意义。结论:rAAV2载体可以介导EGFP 基因高效稳定转染兔结膜上皮细胞,并且对细胞增殖无影响。

Transfection of rabbit conjunctival epithelial cells with adeno-associated virus mediated enhanced green fluorescent protein

Objective:To transfect rabbit conjunctival epithelial cells in vitro with recombinant adeno-associated virus 2-mediated enhanced green fluorescent protein (rAAV2-EGFP),so as to lay a foundation for studying the proliferation and differentiation of conjunctival epithelial stem cells.Methods: Rabbit conjunctival epithelial cells were cultured in vitro.The second passage of rabbit conjunctival epithelial cells were transfected with rAAV2-EGFP in various multiplicity of infection (MOI=104,105,106).The expression of green fluorescent protein was examined 1,3,5,and 7 days after transfection and the transfection rate was calculated.MTT assay was used to study the influence of rAAV2-EGFP on the growth of cells.Results: With the increase of MOI value and the prolongation of transfection,the expression of EGFP was gradually increased and reached its peak on 7-8 days after transfection.MTT assay showed that there was no significant difference between the MOI group and the control group.Conclusion: rAAV2-EGFP can efficiently transfect rabbit conjunctival epithelial cells and have no influence on the proliferation of the cells.