【作者】 熊思齐；

【导师】 夏晓波；

【作者基本信息】 中南大学 ， 眼科学， 2009， 博士

【摘要】 目的探讨氧诱导增殖性视网膜病变小鼠视网膜组织中促红细胞生成素(EPO)mRNA及蛋白质表达的动态变化及意义。方法76只出生后7天(P7)的C57BL/6J小鼠随机分为模型组(70只)及对照组(6只),出生后第7天(P7)模型组小鼠和母鼠一起放入氧气含量为(75±5)%的饲养箱中饲养,出生后第12天(P12)回到正常大气环境中饲养。模型组及对照组在第17天(P17)分别取小鼠6只,FITC-Dextran左心室灌注后行视网膜铺片,荧光显微镜下观察血管分布和形态(每组各3只)。组织学切片观察突破视网膜内界膜的新生血管内皮细胞核数量(每组各3只)。提取模型组小鼠出生后第12(P12)、12.5(P12.5)、13(P13)、13.5(P13.5)、14(P14)、17(P17)、19(P19)、26天(P26)八个时间点的视网膜总RNA及蛋白质,通过RT-PCR及免疫印Western blot技术检测视网膜组织中EPO表达的动态变化趋势。结果荧光造影结果显示模型组小鼠在出生后第17天时视网膜有大量新生血管形成,视盘周围见大范围无灌注区。组织切片可见大量突破视网膜内界膜的新生血管内皮细胞核;模型鼠视网膜组织中EPOmRNA及蛋白质表达水平在缺氧状态下迅速升高,于缺氧48h时到达高峰,然后缓慢下降。结论EPO mRNA及蛋白质表达水平在氧诱导增殖性视网膜病变小鼠视网膜中明显上调,其变化趋势与视网膜新生血管形成相对应。目的筛选能有效抑制EPO表达的小干扰RNA(siRNA)并观察靶向EPO的siRNA在正常氧及低氧环境下对EPO表达的抑制效率。方法设计并合成3条靶向EPO的siRNA,通过阳离子脂质体包裹转染NIH/3T3细胞(转染浓度为100nmol/L)。转染后24小时,采用RT-PCR及Western blot技术检测EPO siRNA对细胞内EPOmRNA及蛋白质表达的抑制效果,从而筛选出能有效抑制EPO表达的siRNA序列。然后将NIH/3T3细胞分成正常氧培养组和低氧培养组,将EPO siRNA转染两组细胞。转染后24小时,采用RT-PCR及Western blot技术观察正常氧及低氧环境下EPO siRNA对细胞内EPO表达的抑制效果。结果设计的3条靶向EPO的siRNA(siEPO1-3)均能不同程度的抑制细胞中EPO的表达。其中,siEPO2对细胞中EPO表达的抑制效率最高,抑制效率达到80%。正常氧及低氧环境下,EPO siRNA能明显抑制NIH/3T3细胞内EPO mRNA及蛋白质的表达。结论正常氧及缺氧环境下,siEPO2能有效抑制EPO的表达,为体内试验提供基础。目的观察促红细胞生成素小干扰RNA(siRNA)对视网膜新生血管形成的抑制作用,进一步阐明促红细胞生成素在视网膜新生血管形成中的调控作用。方法将33只鼠龄为7天的C57BL/6J小鼠置于浓度为(75±5)%高氧环境中生活5天,然后返回正常氧环境中。22只小鼠于出氧舱后当日一只眼玻璃体腔内注射1ul脂质体-EPO siRNA(5ug)混合物,对侧眼注射1ul脂质体-阴性对照siRNA(5ug)混合物。11只不作注射的高氧诱导小鼠作为模型组。另取11只小鼠于正常氧环境下饲养作为正常对照组。FITC-dextran左心室造影视网膜铺片了解视网膜血管形态的改变;组织学切片观察突破视网膜内界膜的血管内皮细胞核数量。采用RT-PCR、Western blot检测视网膜中EPO mRNA及蛋白质的表达水平。结果模型鼠玻璃体腔内注射siEPO2后,视网膜铺片结果显示:siEPO2注射组新生血管芽及荧光素渗漏现象较模型组明显减少;组织学切片结果表明:与模型组相比较,siEPO2注射组突破视网膜内界膜的血管内皮细胞核数量明显减少。玻璃体腔注射siEPO2后,视网膜组织中的EPO mRNA及蛋白质表达水平较模型组明显下调,差异有统计学意义(P<0.05)。结论EPO小干扰RNA可有效抑制视网膜新生血管的形成,进一步证明了EPO在视网膜新生血管形成中所起的调控作用。同时为血管增生性视网膜病变的治疗提供了新的途径。更多还原

【Abstract】 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.更多还原