The Study On The Role Of Erythropoietin In Retinal Angiogenesis

Objective:To explore the changes and signifcance of expression of erythropoietin(EPO) mRNA and protein in mice with oxygen-induced proliferative retinopathy.Methods:Seventy-six 7-day-old C57BL/6J mice were randomly divided into model group(70 mice)and control group(6 mice).On the 7th day after the birth,the mice in model group were raised in fl box full of (75±5)%and then under the normal atmosphere condition on the 12th postnatal day.The mice in control group were raised under the normal circumstance all the time.On the 17th day after birth,6 mice in each group underwent fluorescein perfusion and histological analysis.The expression of EPO,in model group were detected by reverse transcription-polymerase chain reaction and western blot on the 12th, 12.5th,13th,13.5th,14th,17th,19th,26th postnatal day. Results:On the 17th day after the birth in the model group,retinal neovascularization and non-perfused area around the optic post were observed;the expression of EPO mRNA and protein increased 12 hours after hypoxia,reached the peak at the 48 hours,and then decreased slowly:Conclusion:The expressions of EPO mRNA and protein in mice with oxygen-induced proliferative retinopathy is related to retinal anigogenesis. Chapter2 pick out effective small interfering RNA (siRNA)targeting EPOPurpose:To pick out the small interfering RNA(siRNA) which could most effectively inhibit expression of EPO in vitro and investigate the inhibitory effect of siRNA on the expression of erythropoietin in cultured cells under normoxia and hypoxia condition.Method:Three siRNA against erythropoietin were designed and synthesized.Then it was transfected to NIH/3T3 cells by liposome. RT-PCR and Western blot were used to evaluate the efficacy of siRNA on attanuating erythropoietin expression in the cells,The most effective siRNA could be picked out by this means.Then,NIH/3T3 cells were divided into normoxia and hypoxia group.After transfection of erythropoietin siRNA into NIH/3T3 cells by Lipofectamine 2000, erythropoietin expression was examined by using RT-PCR and Western blot.Result:three designed siRNA could suppress EPO expression with different ability,siEPO2 was the most efficient one which could inhibit 80%erythropoietin expression in cultured cell.Meanwhile, erythropoietin mRNA and protein expression in cultured NIH/3T3 cells under both normoxia and hypoxia condition was down-regulated significantly by siEPO2.Conclusion:siRNA against erythropoietin effectively inhibits the expression of erythropoietin in vitor and provides basis for in vivo study. Objective:To observe the effect of inhibition of retinal neovascularization by small interference RNA(siRNA) targeting erythropoietin(EPO).Method:One week old C57BL/6J mice were exposed to(75±2)% oxygen for 5 days,then it returned to the room air to induce retinal neovascularization,siRNA which manifested as most powerful in reducing EPO expression in vitro was intrvitreal injected in the treatment group.Retinal neovascularization was evaluated by angiography with injection of fluorescein dextran and quantification of neovascular proliferative retinopathy after 5 days in room air.Moreover,RT-PCR, immunoblot analysis were used to determined whether local administration of EPO siRNA could affect the expression of EPO in murine retinas.Result:In murine model of oxygen-induced retlnopathy,retinal neovascularization in the eyes with siEPO2 injection was significantly reduced compared with that of the contralateral control eyes.Similarly, histological analysis indicates that neovascular nuclei protruding into viteous cavity was decreased compared to the control eyes.Furthermore, the expression of EPO in the retinas with injection of siEPO2 was drmatically decreased.Conclusion:siRNA against Erythropoietin could inhibit the experimental retinal neovascularization through reducing EPO expression in the retinas of mice.It may provide a powerful and novel therapeutic tool for ischemic-induced retinal diseases.