慢病毒载体介导增强型绿色荧光蛋白两种途径体内转染大鼠角膜基质细胞有效性的试验研究

Study on the effectiveness of Lentiviral vector-mediated enhanced green fluorescent protein transfection in vivo via two ways of corneal stromal cells in rats

目的:观察慢病毒载体介导的增强型绿色荧光蛋白(lenti-EGFP)通过两种途径体内转染大鼠角膜基质细胞的有效性,为角膜疾病尤其是准分子激光术后角膜Haze形成的基因治疗提供实验基础。方法:Lenti-EGFP通过角膜基质注射和刮除角膜上皮敷贴带lenti-EGFP的棉片两种不同方法体内转染大鼠角膜基质细胞,转染后定期裂隙灯观察,进行眼前节照相,以3d、7d、26d、48d四个不同时间点收集角膜,固定后切片,共聚焦显微镜观察。结果:两组转染了lenti-EGFP的角膜2d后增强型绿色荧光蛋白(EGFP)开始表达,7d时达到高峰,26d时表达明显减弱,48d时仅有微弱表达,空质粒组和对照组的角膜基质细胞在各时间段均未见荧光表达。结论:Lenti-EGFP通过上述两种转染途径均可有效的转染角膜基质细胞。

Objective：To observe the lentiviral vector-mediated enhanced green fluorescent protein（lenti-EGFP） in two ways in vivo rat corneal stromal cells transfected with the effectiveness.To provide experimental basis for gene therapy of corneal diseases,in particular the formation of excimer laser corneal Haze.Methods： Lenti-EGFP transfect into tat corneal stromal cells through injected into the corneal stroma and application a piece of cotton with Lenti-EGFP after erasion epithelium,slit-lamp observation regular after transfection,the anterior segment photography,collected the cornea on four different time points（3d,7d,26d,48d）,fixed sections then observation by confocal microscope.Results： After 2d the corneal Transfected of the lenti-EGFP,enhanced green fluorescent protein（EGFP） begin to express,7d reached the peak,26d when the expression was significantly reduced,48d when the only weak expression,empty vector group and control group corneal stromal cells in each time period were no fluorescent expression.Conclusion： Lenti-EGFP transfection by means of the two can be effectively transfected corneal stromal cells.