The Research Of VEGF Small Interfering RNA Inhibiting Retinal Neovascularization

ObjectiveThe retinal neovascular disease,such as diabetic retinopathy,ischemia retinalvein occlusion or retinopathy of prematurity,can cause retinal neovascularization.The vascular endothelial growth factor (VEGF) is the main growth factor whichinduced neovascularization in retina.We use RNA interference technique to inhibitVEGF.We construct VEGF small interfering RNA (VEGFsiRNA) recombinantplasmid and investigate the effect of VEGFsiRNA on the inhibition of retinalneovascularization in mice of oxygen induced retinopathy (OIR) and evaluate theefficacy of VEGFsiRNA in retinal neovascular disease.MethodsThis study included 4 parts:1.We design VEGFsiRNA of monkey and mouse and clone VEGFsiRNA intothe eukaryotic expression vector pSilencer2.1-U6neo to construct the recombinantplasmid.After transformed into E.coli cells,the recombinant plasmid were confirmedby restrict enzyme digestion and sequence analysis.2.Monkey microvascular endothelial cells were cultured in vitro and dividedinto normoxia group and hypoxia group (Cocl₂).Hyoxia group was divided intocontrol hypoxia group,vector group,VEGFsiRNA transfection group randomly.Thecells were transfected with vector plasmids (vector group),VEGFsiRNA recombinantplasmid (VEGFsiRNA group) by the Lipofectamine^TM2000(LF2000) method.Notransfection were performed in the normoxia group and control hypoxia group.Thecell proliferation were detected by MTT methods.The expression of VEGFmRNAand protein were detected by real-time RT-PCR and western blot.3.Establish oxygen induced retinopathy (OIR) mouse (C57BL/6J) model withimproved Smith methods.HE stain and Fluorescein-Dextran angiography of retinalvascular were performed to observe the formation of retinal neovascularization.Dynamic expression of VEGF was detected by real-time RT-PCR. 4.The mice (C57BL/6J) were divided into normoxia group and hypoxia controlgroup,vector group and gene therapy group randomly.The vector group were the P12model mice injected with vector plasmids.The gene therapy group were the P12model mice injected with VEGFsiRNA recombinant plasmids.The all plasmids weremediated by Lipofectamine^TM2000(LF2000).Results1.The recombinant plasmids were confirmed by enzyme digestion and sequenceanalysis.The pasmids were successfully constructed.2.The cell growth curve of 24h,48h,72h showed that the growth rate becameslower in cells of VEGFsiRNA group.The expression of VEGFmRNA and proteinwere faint in the normoxia group,but increased obviously after hypoxia.Theexpression of VEGFmRNA and protein in VEGFsiRNA group were decreasedcompared with hypoxia control group.VEGFmRNA and protein levels were reducedby 48.07%,51.82%.3.The retinal neovascularization of oxygen induced retinopathy (OIR) mouse(C57BL/6J) model appeared in P14 and peaked in P17 and then gradually declined.The VEGFmRNA expression was faint in P12.It reached peak in P14 and thendeclined gradually.4.There were few vascular endothelial cell nuclei extending beyond the internallimiting membrane(ILM)in the normal group,while a large number of vascularendothelial cell nuclei in the control group and vector group were extending into ILM,incidence rate 100%.There is significant difference in the number of nuclei extendingbeyond the ILM between gene therapy group and the control group,the vector group.Immunofluorescence showed the expression of VEGF was weakly positive innormoxia group and strong positive in hypoxia control group and vector group.TheVEGF significantly reduced in gene therapy group.The expression of VEGFmRNAwas faint in the normoxia group,but increased obviously after hypoxia.Theexpression of VEGFmRNA in gene therapy group were decreased compared withhypoxia control group.VEGFmRNA level was reduced by 43.39%. Conclusion1.In vitro:pSilencer2.1-U6neo-VEGFsiRNA mediated Lipofectamine^TM2000can effectively inhibit the growth rate of the monkey retinal microvascularendothelial cells and the expression of VEGFmRNA and protein.2.In vivo:Retinal neovascularization can be efficiently inhibited bypSilencer2.1-U6neo-VEGFsiRNA mediated Lipo fectamine^TM2000 throughintravitreal injection.