| 1. Objective:Proliferative vitreoretinopathy (PVR) is the major complication of retinal detachment surgery and the main reason for its failure.It often lead to serious visual impairment. vitrectomy in the development of the past 30 years has greatly changed the treatment of pathologic appearance, the application of various techniques such as retinal surgery incision and silicone oil almost make all kinds of complex retinal detachment anatomic replacement, but vision rehabilitation of most patients after surgery is not satisfaction. PVR relapse after surgery also affected the outcome. Surgery has rare influence against neuronal cell death and cell proliferation. In the same way the result is not satisfactory for PVR drug therapy. Toxicity of medicine restricts its clinical application. Therefore to find new ways to control PVR is imperative. Gene therapy is a new technique commence from 1990s opening up a new way of modern therapy.Pigment epithelium-derived factor (PEDF) is a recently discovered protein, with a variety of biological activity has been widespread concern in recent years. It can affect the retina division, development and maturation as a neuroprotective factor and participation in the regulation of angiogenesis in the developmental and physiological status of the body play an important role. Experimental studies have found PEDF can increas pigment particles of newborn rats'cultured RPE cells show that PEDF have the role of regulating RPE cells differentiation. Lack of PEDF can lead to RPE cells degradation.PEDF can completely inhibit microglia cell division induced by colony stimulating factor. By changing the shape of microglial cells turn it into inactive status. Then indirectly inhibit the proliferation of astrocytes. PEDF can inhibit cell proliferation of macrophages and inflammatory response induced by macrophages.PEDF can also increase expression FasL in endothelial cells, FasL-induced leukocyte apoptosis on the vascular wall, inhibiting leukocyte extravasation.Ogata found the concentration of PEDF is lower in the case of PVR than in the case of rhegmatogenous retinal detachment. Show PEDF may has the function of inhibit PVR ,but its mechanism is not yet clear. They deduce it may because PEDF inhibit the activity of VEGF in promoting growth and migration. PEDF is also a antioxidant, which can inhibit vascular endothelial cells reactive oxygen (ROS),then inhibit VEGF expression.Currently, the research on the role of PEDF in PVR is limited. Scholars only mensurated PEDF concentration in vitreous humor of PVR vases.The role of PEDF in PVR has not been experiment. Accordingly, our experiment constructed PEDF eukaryotic expression vector. In vitro studies, we observed PEDF gene-modified RPE cells inhibition to proliferation induced by macrophage conditioned medium. We also observed the restraining role of PEDF transfered by Vitreous cavity injection in PVR model rats induced by macrophages. 2. contentFirst construction recombinant plasmid pcDNA3-PEDF, PEDF gene were cloned into the eukaryotic expression vector pcDNA3 from pGEM-T-PEDF plasmid. Application digestion and DNA sequencing. pcDNA3-PEDF eukaryotic expression vector transfered RPE cells by Liposome. After G418 selection, MTT assay induced in human macrophage conditioned medium to observe transfected RPE proliferation. Then by injection into the vitreous cavity of PVR model rat eyes, study of the expression in the eyes and the the restraining role to PVR.The results show : We successfully constructed of eukaryotic expression vector pcDNA3-PEDF. The plasmid was identified and sequencing results show that PEDF in which the presence of the gene sequence were identical in Genebank. Transfered RPE cells by Liposome expressed PEDF protein stablely,which can be detected through immunocytochemistry techniques. MTT assay show after transfection of RPE cells can inhibit the proliferation induced by macrophage conditioned medium.Liposome-mediated pcDNA3-PEDF injected in rats'vitreous cavity can be seen stable expression in the eyes, PVR induced by macrophages also significantly be inhibited.3. Major achievements(1) We first experimentally observed human RPE cells proliferation in macrophage conditioned medium after PEDF gene transfered. Concluded PEDF can inhibit macrophage conditioned media induced RPE cell proliferation. (2) We first experimentally observed liposome-mediated eukaryotic expression vector pcDNA3-PEDF injected in rats'vitreous cavity can be seen stable expression in the eyes, and PVR induced by macrophages can significantly be inhibited.4. The research achievements, significance and value:Since 1989, scholars had done a lot of research on the role of PEDF. PEDF as a biological, endogenous soluble cytokines , neurotrophic and protective factor with good histocompatibility has the powerful role of inhibit angiogenesis. The experiment proved that adenovirus, retrovirus carriers PEDF gene can be transferred to the target cells, retinal intravitreal injection rAAV-PEDF may increase PEDF protein content in eyes significantly, and can keep more time to play effect, have broad clinical application.However, the role of PEDF in PVR is not clear. Although detecting vitreous humor in PVR cases found PEDF decrease which indirectly proved PEDF related to the formation of PVR. But the role of PEDF in PVR has not been experiment. We first experimentally concluded PEDF can inhibit macrophage conditioned media induced RPE cell proliferation. And PEDF gene was transferred in eyes could also inhibit PVR induced by macrophages significantly. So this experiment laid the foundation for further examine PEDF roles in PVR and its application in the PVR prevention.
|
PDF Full Text Request