The Study On Inhibition Of Proliferative Vitreoretinopathy With Adenovirus-mediated Delivery Of P21WAF1/CIP1

ObjectiveThe purpose of this study is to establish a rabbit proliferative vitreoretinopathy (PVR) animal model and investigate the inhibition effect for PVR by using adenovirus-mediated p21WAF1/CIPt vectors.Methods1. The establishment of PVR animal model:twenty-two pigment rabbits were randomly divided into two groups:experimental group and control group. Each group had28eyes of14rabbits. In experimental group, the human platelet-rich plasma (PRP) intravitreal injection combined cryotherapy operation had been used to establish PVR animal model and the rabbit PRP intravitreal injection operation had been used in control group. The indirect ophthalmoscopy, B scan and HE staining technique were used to investigate the successful rate of rabbit PVR model.2. The role of p21WAF1/CIP1gene in PVR pathogenesis:twenty-seven pigment rabbits were randomly divided into two groups, three rabbits in the normal group and24in the experimental group. In experimental group, each rabbit was randomly selected single eye as the experimental eye. Rabbits in normal group did not receive any operation and rabbits in experiment group were operated with human platelet-rich plasma (PRP) intravitreal injection combined cryotherapy. Western blot and RT-PCR methods had been used to measure p21WAF1/CIP1、CDK2、cyclin E protein and mRNA expression in retinal tissue at7d^14d^21d and28d after operation.3. In vitro:The cultured human retinal pigment epithelial cells407lines (hRPE407) were divided into three groups:phosphate buffered saline (PBS) group, negative control (Ad-NC) group and adenovirus p21WAF1/CIP1gene vector (Ad-p21) group. Western blot and RT-PCR methods were used to detect the level of p21WAF1/CIP1, CDK2and cyclin E in three groups, and MTT method to observe cell proliferation, flow cytometry to detect the cell cycle and Transwell chamber to measure cells migration. 4. In vivo:forty pigment rabbits were randomly divided into four groups which included normal group (untreated), PBS group (intravitreal injection of PBS), Ad-NC group (intravitreal injection of adenovirus empty vector) and Ad-p21group (intravitreal injection of adenovirus-mediated p21WAF1/CIP1gene vector). There were2rabbits in the normal group and16rabbits in each other groups. Each rabbit was randomly selected one eye as the experimental eye except the rabbits in normal group. The human PRP intravitreal injection combined cryotherapy method had been used to establish PVR animal model. At third day after operation, the experimental eyes received different intravitreal injection treatments according to different groups. Western blot and RT-PCR method had been used to detect expression of p21WAF1/CIP1, CDK2and cyclin E in retinal tissues at14th day. The rate of retinal detachment had been calculated at28th day with indirect ophthalmoscopy, B-scan and HE staining methods.Results1. At28th day after operation, the number of eyes with retinal detachment was20/20(100%) in experimental group and13/20(65%) in control group. The difference in the frequence of retinal detachment between the two groups was statistically significant (P<0.01).2. The results of Western blot and RT-PCR showed that p21WAF1/CIP protein and mRNA expression in retinal tissues were lower in experimental group than those in normal group (P<0.01), and the lowest level was presented at14th day after operation. The expression of CDK2and cyclin E were higher in experimental group than those in normal group (P<0.01) and the highest level was appeared at14th day after operation.3. Western blot and RT-PCR results showed that p21WAF1/CIP protein and mRNA expression were enhanced in rabbits with p21WAF1/CIP gene transfection. The inhibition rate of cell proliferation reached to43.13%at48h after cells transfected with p21WAH/CIP gene. The percentage of cells at the G0/G1phase determined by flow cytometry was significantly higher in Ad-p21group than those in the PBS and Ad-NC group(P<0.01). The number of cells migrated through Transwell chamber upper layer was significantly lower in Ad-p21group compared to PBS and Ad-NC group (P<0.01).4. Indirect ophthalmoscopy and B-scan results showed that there were12/12(100%) eyes,11/12(91.67%) eyes and4/12(33.33%) eyes with retinal detachment in PBS group, Ad-NC group and Ad-p21group respectively at the28th day after operation. At the14th day, the protein and mRNA expression of p21WAF1/CIP in retinal tissues were significantly higher in Ad-p21group than those in normal group, PBS group and AD-NC group (P<0.01). However, the levels of CDK2and cyclin E were significantly lower in this group compared to other groups(P<0.01).Conclusion1. Intravitreal injection of human PRP combined cryotherapy operation successfully established PVR animal models and the occurrence of retinal detachment rate was100%.2. p21WAF1/CIP1gene may play an important role in PVR pathogenesis.3. The p21WAF1/CIP gene mediated by adenovirus can effectively inhibit the proliferation and migration in cultured human retinal pigment epithelial cells.4. The adenovirus-mediated p21WAF1/CIP gene vector intravitreal injection can significantly reduce the rate of retinal detachment.