腺病毒与慢病毒载体转染离体兔角膜基质细胞的有效性对比研究

目的观察比较腺病毒载体(adenovirus vector,AV)与慢病毒载体(lentivirus vector,LV)分别介导增强型绿色荧光蛋白(enhanced green fluorescent proteins,EGFP)对体外培养的兔角膜基质细胞(rabbit corneal stroma cells,RCSC)的转染效率,探讨较优一种病毒载体作为角膜基因治疗载体的可行性。方法行离体RCSC原代、传代培养及鉴定;实验分为转染组(AV-EGFP转染组和LV-EGFP转染组)和对照组,观察转染组在不同感染复数(multiplicity of infection,MOI)及感染后24h、48h、72h在倒置荧光显微镜下EGFP的表达情况,流式细胞技术检测转染效率。结果 AV-EGFP转染组感染RCSC后从24～48h就开始可见明显的绿色荧光,起始时间早于LV-EGFP转染组(48h)。LV-EGFP转染组在MOI≤1000时,随着MOI值增大,检测到的细胞转染率逐渐增大,两两比较差异均有统计学意义(均为P<0.05);AV-EGFP转染组在MOI>10时,随着MOI值增大,检测到的细胞转染率逐渐增大,两两比较差异均有统计学意义(均为P<0.05);而在同一MOI值下(MOI>1),LV-EGFP较AV-EGFP转染效率更高,差异均有统计学意义(均为P<0.05)。结论 AV与LV均可有效转染离体RCSCs同一MOI值下(MOI>1),LV转染效率更高。

Efficiency comparison of gene transfer into the isolated rabbit corneal stoma cells in vitro using adenovirus and lentivirus vector

Objective To observe the transfection efficiency of adenovirus vector mediated enhanced green fluorescent proteins(AV-EGFP) and lentivirus vector mediated enhanced green fluorescent proteins(LV-EGFP) in the isolated rabbit corneal stromal cells in vitro,and to investigate the feasibility of the better virus vector as one of the gene therapy vector for corneal disease.Methods The primary cultured and subcultured keratocytes were identified from New Zealand white rabbits and cells identification.The cells were exposed to different multiplicity of infection(MOI) of LV-EGFP and AV-EGFP in transfective groups and the same quality of blank culture medium in contrast group.We observed the expression of EGFP by inverted fluorescence microscope at 24 hours,48 hours and 72 hours and then calculated the transfection efficiency through flow cytometer.Results AV-EGFP transfected rabbit corneal stromal cells for 24 hours to 48 hours green fluorescence can be seen clearly from the beginning,and earlier than LV-EGFP(48 hours).MOI as or less than 1000,with the increase of MOI,cells transfection efficiency was gradually increase in LV-EGFP group,and there was statistical difference(all P<0.05).MOI more than 10,cells transfection efficiency was gradually increased in AV-EGFP group,and there were statistical differences(all P<0.05).At the same MOI(MOI>1),the transfection efficiency of LV-EGFP was specially higher than AV-EGFP and the difference was statistically significant(all P<0.05).Conclusions Adenovirus and the Lentivirus vector can be efficient in vitro transfecting rabbit corneal stromal cells,and Lentivirus vector transduction efficiency was higher than Adenovirus at the same MOI.