Studies On Experimental Choroidal Neovascularization Inhibited By Endostatin Genetherapy

PartⅠ Experimental choroidal neovascularization is inhibited by subretinal administration of recombination Endostatin proteinObjective Endostatin is an endogeneous angiogenesis inhibitor.The purpose of this study was to investigate the inhibiting effect of Endostatin on experimential choroidal neovascularization (CNV) in the eyes of rats. Methods Experimental CNV was induced by laser photocagulation . The animals were given subretinal injections of recombinant human Endostatin 20ul(5ug/ul) or 0.9% chlorine sodium .The intensity of fluorescein leakage from the photocoagulated lesions was studied 13days after photocoagulation. The area of CNV at each site was measured using high molecular weight FITC-dextran (MW2χ106)with high resolution angiography in RPE-choroid –sclera flat mounts.In addition,8 eyes in each group were removed and fixed 14 days after photocoagulation, cut into thin sections. The sections were examined by light microscopy. Immunolocalization of Endoglin(CD105) and factor VШof CNV lesions on sections was studied by immunohistochemical evaluation.Results After Endostatin injection,fluorescein leakage from the CNV lesions decreased significantly compared with the control eyes.The average area of CNV at sites of the Bruch's membrane rupture showed significant difference between the eyes injected with Endostatin and the control eyes .Endothelial cells demonstrated strong immunoreactivity of CD105 and factor VШ in CNV lesions of control eyes.No CD105-positive cell was not detected in normal chorioretinal tissues.Conclusions The development of CNV can be inhibited by injection of Endostatin, which suggests that Endostatin may be benefical in treating CNV and that further studies can be considered to evaluate this possibility. PartⅡ The Culture of Brown Norway Rat Retinal Pigment Epithelial Cells Objective To investigate the effective methods of isolation and culture of rat retinal pigment epithelial cells. Methods Whole eyes of 15 days Brown Norway (BN)rats were treated with hyaluronidase and collagenase followed by trypsin, these enzymes release sheets of RPE from adherent attachments to the retina and choroids.Trypsin was then used to dissociate the sheets into single cells.Primary cells'plating rate, successful culture rate,doubling time(DOT) were calculated.The identification and ultrastructure of cells were also observed .Result This technique give high cell yields and high viability.Cultures reached confluency in short times.Light and electron microscopy demonstrated that most of the normal morphological characteristics of rat RPE cells in vivo were maintained in vitro.Conclution The rat RPE cells obtained by the two steps enzyme gradation digestion may be useful in studies of RPE in vitro. Part Ⅲ Transduction and expression of Endostatin gene in Brown Norway Rat Retinal Pigment Epithelial Cells by gene transfer procedures in vitroObjective To determine the feasibility of liposome or electropulse-mediated Endostatin gene transfer to cultured Brown Norway Rat Retinal Pigment Epithelial Cells. Methods Cultured brown norway rat retinal pigment epithelial Cells were infected with the recombinant eukaryotic expression plasmid pSecTagA-hEndostatin by LipofectamineTM2000(LF2000)or electropulse. The positively expressed cell were selected with zeocin .The production of Endostatin was evaluated by PCR, Hybridization in situ ,western bloting and immunohistochemistry.The secretion of Endostatin produced by RPE cells was quantified by an enzyme-linked immunosorbent assay(ELISA).The inhibition effect of the expressed protein on the human umbilical vein endothelial cells (ECV304),K562 and RPE cells were also evaluated by MTT.Result LF2000 or electropulse-mediated gene transfer can introduce Endostatin gene into cultured RPE.Gene expression were revealed by PCR, Hybridization in situ ,western bloting , immunohistochemistry and ELISA.MTT showed 50% inhibition concentration of the Endostatin protein on ECV304 was45.68 ug/ml.But it had no inhibition effect o...