Inhibitory Effect Of Lentiviral-mediated Small Hairpin RNA Targeting Netrin-1on Retinal Neovascularization

Chapter1Relationship between netrin-1and vascular endothelial growth factor in proliferative diabetic retinopathyPurpose:To investigate the significance of netrin-1in the pathogenesis of proliferative diabetic retinopathy (PDR) and determine the correlation between levels of netrin-1and VEGF in the vitreous fluid of PDR patients.Methods:A total of18eyes from18patients were included in our study.10of them were PDR patients and8were NDRP patients. Undiluted vitreous fluid samples were collected during pars plana vitrectomy, and appropriate blood samples were collected. Netrin-1and VEGF levels in the vitreous fluid and plasma were determined by Enzyme-linked Immunosorbent Assays.Result:The levels of netrin-1and VEGF in the vitreous of PDR patients were significantly higher than that in the controls (Mediannetrin-1=509.94±138.85vs.85.91±11.72pg/ml, p＜0.001and MedianVEGF=762.60±143.14vs.77.52±7.75pg/ml, p<0.001). However, up-regulations of netrin-1and VEGF were not correlated (rho=0.200; P=0.704).Conclusions:Netrin-1and VEGF were independently elevated in vitreous fluid of the PDR patients. Therefore, netrin-1may play a role additional to VEGF in angiogenesis and maybe a potential therapeutic target for PDR.Chapter2Construction and package of lentiviral-mediated netrin-1small hairpin RNA and screened it by transfection in vitroPurpose:To Construct the lentiviral-mediated netrin-1small hairpin RNA(shRNA) and scramble shRNA.Methods:Lentivirus carriers were constructed which inherited netrin-1shRNA, and then co-transfected to the293T cells with the pHelper1.0and pHelper2.0carrrier,8h later, they were cultured with completed culture medium, and48h later, lentiviral particles in the mediums were harvested and concentrated. The bEnd.3cells were transfected with lentivirus particles to determine its transfection rates, total protein were extracted72h later, netrin-1was examined by western-blot.Result:Compared with the scramble shRNA, netrin-1shRNA reduced the netrin-1expression in bEnd.3cells by75.45%successfully.Conclusion:The lentiviral-mediated netrin-1shRNA we constructed can transfect the bEnd.3cells efficiently and suppress netrin-1successfully.Chapter3Suppression of the retinal neovascularization by lentiviral-mediated netrin-1small hairpin RNAPurpose:To investigate netrin-1expression in the retinas of oxygen induced retinopathy (OIR) mice and determine its role in retinal neovascularization by knocking down netrin-1in the retinas of OIR mice.Methods:One week old C57BL/6J mice were exposed to(75±2)%oxygen for5days, and then returned to the room air to induce retinal neovasculariza-tion. Lentiviral mediated netrin-1shRNA which were efficient in reducing netrin-1expression in vitro were intravitreal injected in the treatment group, while lentiviral mediated scramble shRNA were administered intravitreally as control. Real time PCR, and western-blot were used to detect the netrin-1expression in OIR mice retina. Retinal neovascularization was evaluated by angiography with injection of FITC-dextran and quantification of neovascular proliferative retinopathy after7days in room air.Results:Both mRNA and protein expression of netrin-1were significantly up-regulated in the P17OIR mice retinas. Treatment of OIR mice with specific lentivirus-mediated netrin-1shRNA dramatically reduced neovascular tufts and non-perfuse areas in the retina.The mean neovascular vessels outgrew the innerlimit membrane in the treatment group were9.8±1.7, compared to the control group were20.3±3.4, which reduced nearly50%neovasculars.Conclusions:Netrin-1was highly expressed in the retina under ischemic conditions and lentiviral mediated netrin-1shRNA could reduce pathological retinal neovascularization effectively, which supplied a new target in the therapy of neovascular related retinopathy.