Study On Lentiviral Vector-Mediated Enhanced-Green Fluorescent Protein Via Two Ways Transfection In Vivo Of Corneal Stromal Cells In Rats

Objective: To observe on the effectiveness and safety of Lentiviral vector-mediated enhanced green fluorescent protein transfection in vivo via two ways of corneal stromal cells in rats.To explore a transfected with vector and means of simple,effective and safe , providing an experimental basis for gene therapy of corneal diseases.Methods: Lenti-EGFP by two different methods transfection in vivo of corneal stromal cells in rats, channels 1 Lenti-EGFP injected into corneal stromal cells in rats through the Micro injector; channels 2 scraping corneal epithelial, sticking cotton sheet which with equal Lenti-EGFP virus solution. After transfection, Regular observations with slit lamp, observation on expression of green fluorescent and external eyes. Collecting cornea at 3d, 7d, 26d, 48d, fixed sections,the HE staining,to observat the general form of layers of cells in the cornea; Observed and photographed by confocal microscopy, to understand the expression of corneal fluorescence intensity, duration, and distribution;by electron microscopy observed the changes in cell ultrastructure; detection of apoptosis, underst anding corneal cell apoptosis in experimental group and control group.Results: 1.Via slit lamp Quran light seeing the cornea is blue-green which transfected the lenti-EGFP,no fluorescent expression in normal group corneas. 2.HE staining showed that the layers of the cornea of each group of rats with no obvious morphological abnormalities, Seen by electron microscopy, ultrastructure of corneal stromal cells the experimental group no obvious abnormalities.3. Confocal microscope seeing: corneal stromal cells transfected lenti-EGFP , the enhanced green fluorescent protein (EGFP) begin to express after 2d, at 7d the Fluorescence expression reached the peak, the whole layer of the cornea are visible fluorescence expression, 26d when the expression was significantly decreased, 48d when there is only a slight expression of epithelial; empty plasmid group at 3d and 7d, corneal epithelium and stroma layer can be seen weak fluorescence, not in stroma cells,26d and 48d when the fluorescence is no longer expressed; the control group all layer of the cornea in each time period were no fluorescent expression. 4.Tunel detected the apoptosis rate of the experimental group is higher than normal group, but compared with the normal group was no significant difference (P> 0.05). Conclusion: Lenti-EGFP can be transfected corneal stromal cells safly and effectively by both way. The initial transfection, the Transfection efficiency of injection is stronger,with the time the transfection efficiency in two ways quite similar,the injection group is a little higher. The two technologies are both Simple, genetically modified fast, and high transfection efficiency, safety. Provide new ideas to all causes of corneal disease,especially inhibition Haze formation after excimer laser surgerycorneal and inhibition Rejection after Half-layer keratoplasty.