目的:评价8型重组腺相关病毒(rAAV8)介导增强型绿色荧光蛋白基因(EGFP)转染角膜基质细胞后的表达及对细胞增殖的影响。方法:以不同MOI的rAAV8-EGFP转染大鼠角膜基质细胞,转染后以倒置荧光显微镜观察角膜基质细胞中GFP的表达,流式细胞仪分析角膜基质细胞中rAAV8-EGFP表达的阳性率。MTT法分别检测rAAV8和rAAV8-EGFP转染对角膜基质细胞增殖的影响。结果:倒置荧光显微镜观察rAAV8-EGFP转染角膜基质细胞后GFP的阳性表达7d达到高峰,此时流式细胞仪检测rAAV8-EGFP对角膜基质细胞的转染效率分别为31.5%(MOI=5×103),42.5%(MOI=5×104),54.8%(MOI=5×105);MTT检测结果显示rAAV8及rAAV8-EGFP转染对角膜基质细胞增殖均无明显影响。结论:rAAV8-EGFP能有效地转染角膜基质细胞,并且对细胞增殖无明显影响。

·AIM:To evaluate the expression and the effect of recombinant adeno-associated virus(rAAV)vector-8 transduced enhanced green fluorescent protein(EGFP)gene into rat keratocytes.·METHODS:rAAV8-EGFP was transfected respectively into rat keratocytes at different multiplicities of infection.The inverted fluorescent microscope was performed to assess GFP expression and the percentage of positive GFP in the cells was analyzed by flow cytometric analysis.The effects of rAAV8 and rAAV8-EGFP on cell proliferation were assessed by methyl thiazolyl tetrazolium(MTT)method.·RESULTS:The inverted fluorescent microscope was performed to assess GFP expression.The EGFP expression peak occurred at 7d after transfection and the expression level was 31.5%(MOI=5×103),42.5%(MOI=5×104),54.8%(MOI=5×105),respectively.Results of MTT assay indicated there was no inhibitory effect on keratocytes by rAAV8-EGFP and rAAV8 transduction respectively.·CONCLUSION:The rAAV8 vector can deliver effectively EGFP genes into rat keratocytes,and there is no inhibitory effect on keratocyte by rAAV8-EGFP transduction.·

R772.2

中国国家自然基金青年资助项目(No.30901650);中国广东省自然基金资助项目(No.10451051501005773)~~