Experimental Study On Laser Photocoagulation Induced Choroidal Neovascularization Is Inhibited By Gene Tansfection Of PEDF

Objective:1. Recombinant plasmid PEDF-GFP was transduced into the retina of the BN rats using cationic liposome, then observe the expression and location of the PEDF-GFP.2. Transducing PEDF-GFP plasmid to the retina of the BN rats with cationic liposome through different gene delivery routes, to explore the suitable routes of administration.3. To investigate the effect of gene transferction of PEDF-GFP on the eyes of rats with krypton laser induced CNV.Methods:1. 24 BN rats were randomly divided into 6 groups, five groups were subretinal injected with PEDF-GFP gene that mediated by cationic liposome , others were control group, 1day, 1 week, 2 weeks, 3 weeks and 4 weeks after photocoagulation, the expression of GFP was observed under fluorescence microscope, and total RNA was abstracted from the retinal and choroidal tissue, the mRNA of PEDF gene was detected by RT-PCR.2. 24 BN rats were randomly divided into 6 groups, five groups were intraviteal injected with PEDF-GFP gene that mediated by cationic liposome, others was controlrol group, 1day, 1 week, 2 weeks, 3 weeks and 4 weeks after photocoagulation, the expression of GFP was observed under fluorescence microscope, total RNA was abstracted from the retinal and choroidal tissue, and the mRNA of PEDF gene was detected by RT-PCR.3. 48 BN rats after photocoagulation were randomly divided into 3 groups as PEDF group, liposome group and controlrol group. Lipofectamine 2000 andPEDF-GFP plasmids were intraviteal injected in the eyes for PEDF group, pure Lipofectamine 2000 were intraviteal injected in eyes for liposome group, The controlrol eyes were not interfered . The injection was performed at photocoagulation day. lw, 2w, 3w, 4w after photocoagulation, the intensity of fluorescein leakage from the photocoanulated lesions was studied by FFA, The area of CNV at each ruptured site was measured using high molecular weight FITC dextran ( MW 2X106) for high resolution annionraphy in RPE-choroid sclera flat mounts, pathologic examination and PEC AM-1 immunohistochemis-try were performed to evaluate the effect of PEDF on the experimental CNV. Results:1. 1 day after subretinal injection of gene PEDF-GFP into BN rat eyes, Under fluorescent microscope green fluorescence was observed in the retina include RPE cell near the injection site and GFP also can be seen in the distant place. The intensity became stronger on the 2 > 3 weeks than 1 week, and it could sustained 4 weeks. GFP in the control group was negative. The expression of PEDF mRNA was also elevated after transfection lday, achieved peak atK 2weeks and maintained stable 4 weeks after transfection. But the PEDF mRNA expression in the control group was weak .2. 1 day after intraviteal injection of gene PEDF-GFP into BN rat eyes, green fluorescence was emitted from the whole retina include RPE cell under fluorescent microscope.The distribution of GFP was extensive and uniform and not confined around the injection site. The intensity became stronger on the 2^3 weeks than lweek, and it could sustained 4 weeks. GFP in the control group was negative. The expression of PEDF mRNA was also elevated after transfection lday, achieved peak at 1 > 2week and maintained stable 4 weeks after transfection, but the PEDF mRNA expression in the control group was weak.3. 1 week, 2 weeks, 3 weeks and 4 weeks after photocoagulation, the fluorescein leakage all appeared in PEDF group, liposome group and control group, but fluoresecin leakage of PEDF group was less than that of liposome group and control group in every phase, fluoresecin leakage of liposome groupwas similar to control group. Pathologic examination in the control group found retinal edema > neutrophilic leukocytes. pigment macrophages * migratory andproliferative RPE cells . fibrocytes and CNV 1 week after photocoagulation. Retinal edema regressed, neutrophilic leukocytes disappeared, pigment macrophages decreased, fibrocytes increased, RPE cells proliferated and the areas of CNV increased after 2 weeks photocoagulation. Pigment macrophages were not found, fibrocytes and migratory RPE cells more proliferated and a great deal CNV were enwrapped by fibrocytes after 4 weeks photocoagulation. Liposome group was similar to control group. But in the PEDF group, after 2 weeks photocoagulation internal granular layer was disordered, external granular layer disappeared, RPE celluar layer and Bruch's membrane ruptured, superficial layer of choriocapillary was injuried, after 4 weeks photocoagulation RPE cells proliferated and the areas of CNV were less than that of control group. Although immunohistochemistry for PECAM-1 all displayed positive staining in PEDF groups liposome group and control group, positive staining of PEDF group was evidently less than that of control group and liposome group at every phase, positive staining of control group was similar to liposome group. The areas of CNV of PEDF were evidently less than those of control group and liposome group at every phase. There were no statistic differences for the areas of CNV between control group and liposome group. Conclusion:1. Cationic liposome can mediate PEDF-GFP gene into the retina include RPE cell of the BN rat effectively, and their stable expression can maintain 4 weeks after transfection.2. Subretinal injection and intravitreal injection both are effective gene delivery route, intravitreal injection is more convenient.3. PEDF gene trasfection can effectively inhibit CNV in BN rats induced by krypton laser photocoagulation.