1型重组腺相关病毒介导增强型绿色荧光蛋白基因转染人脐静脉内皮细胞的体外研究

目的 评价1型重组腺相关病毒(rAAV1)作为新生血管性角膜病变基因治疗载体的可行性.方法 rAAV1-EGFP按转染倍数(MOI)5×103,5×104,5×105转染ECV304细胞,转染后在倒置荧光显微镜下观察ECV304细胞中GFP阳性表达情况和细胞生长形态特点等,流式细胞仪检测rAAV1-EGFP对EVE304细胞的转染效率.MTT法检测rAAV1-EGFP对EVC304细胞增殖的影响.结果 转染后2 d,MOI=5×105组细胞中的GFP开始表达;表达强度随MOI值的增高而增强,同时GFP的表达强度随着时间延长而逐渐增强,转染后第7天达到高峰,此时流式细胞仪检测rAAV1-EGFP对ECV304细胞的转染率分别为45.90％(MOI=5×103),58.56％(MOI=5×104),68.31％(MOI=5×105).AAV1-EGFP转染ECV304细胞后.细胞生长及形态正常.MTT法显示转染后72、96 h各转染组与未转染组之间A值无显著性差异.结论 rAAV1-EGFP对ECV304细胞转染稳定、有效,重组腺相关病毒对细胞增殖无明显抑制,重组腺相关病毒可作为基因治疗角膜新生血管性病变的载体.

Recombinant type 1 adeno-associated virus-mediated transfection of ECV304 cells with enhanced green fluorescent protein gene in vitro

OBJECTIVE: To assess the feasibility of recombinant type 1 adeno-associated virus (rAAV1) as a vector for gene therapy of corneal neovascularization. METHODS: The rAAV1 vector carrying enhanced green fluorescence protein (EGFP) gene (rAAV1-EGFP) was transfected into ECV304 cells at different multiplicities of infection (MOI=5 x 10(3), 5 x 10(4), 5 x 10(5)). EGFP expression in the cells was observed under inverted fluorescence microscope, and the EGFP-positive cell percentage determined by flow cytometry. MTT assay was used to assess the proliferation of the transfected cells. RESULTS: The cells with rAAV1-EGFP transfection at MOI of 5 x 10(5) began to exhibit GFP expression 2 days after transfection, and the fluorescence intensity increased with the MOI used for transfection. GFP expression reached the maximum on day 7, at the point of which the transduction efficiency of rAAV1-EGFP in ECV304 cells was 45.90%, 58.56% and 68.31% corresponding to MOIs of 5 x 10(3), 5 x 10(4), and 5 x 10(5), respectively. MTT assay did not reveal significant difference in the absorbance between the transfected cells and the control cells at 72 and 96 h after transfection. CONCLUSION:arAAV1-EGFP gene can be stably and efficiently expressed in ECV304 cells without causing cell growth inhibition, suggesting the potential of rAAV1 as a safe and efficient vector for gene therapy of corneal neovascularization.