【作者】 蒋剑；

【导师】 夏晓波；

【作者基本信息】 中南大学 ， 眼科学， 2008， 博士

【摘要】 第一章:HIF-1α和VEGF小干扰RNA对人血管内皮细胞HIF-1αl和VEGF表达的抑制目的:探讨缺氧诱导因子-1α（HIF-1α）和血管内皮生长因子（VEGF）小片段干扰性RNA（siRNA）对人血管内皮细胞HIF-1α和VEGF表达的影响。方法:构建HIF-1αsiRNA重组质粒。将体外培养的人脐静脉血管内皮细胞（HUVEC-12）分成常氧（20%）组和低氧（1%）组。低氧组中,采用脂质体Lipofectamine^TM2000（LF2000）分别转染空载体质粒（空载体组）、HIF-1αsiRNA（HIF-1α组）、VEGF₁₆₅siRNA（VEGF组）和HIF-1αsiRNA+VEGF₁₆₅siRNA（共转染组）,低氧组中未转染的细胞为低氧对照组。常氧组细胞不做转染。采用逆转录-聚合酶链反应（RT-PCR）检测转染后重组质粒的表达;RT-PCR和免疫细胞化学法检测细胞中HIF-1α和VEGF基因和蛋白表达的变化。结果:成功构建HIF-1αsiRNA重组质粒。人脐静脉血管内皮细胞转染24h后,HIF-1αsiRNA和VEGF₁₆₅siRNA重组质粒有表达。细胞HIF-1αmRNA及蛋白表达水平:常氧组细胞HIF-1αmRNA有表达,但未见明显HIF-1α蛋白表达,低氧对照组和空载体组细胞HIF-1αmRNA和蛋白表达较常氧组增强,而HIF-1α组较低氧对照组明显减弱（P＜0.01）,mRNA抑制效率为59.8%。细胞VEGF mRNA及蛋白表达水平:常氧组细胞仅见微弱的VEGF mRNA和蛋白表达,而缺氧后表达明显上调（P＜0.01）;HIF-1α组、VEGF组和共转染组表达较低氧对照组减弱（P＜0.01）,mRNA抑制效率分别为39.7%、68.1%和82.3%,其中共转染组抑制效果最明显。结论:HIF-1α和VEGF₁₆₅siRNA能有效抑制人脐静脉血管内皮细胞HIF-1α和VEGF的表达。第二章:HIF-1a和VEGF小干扰RNA对鼠视网膜新生血管的抑制目的:探讨缺氧诱导因子-1α（HIF-1α）和血管内皮生长因子（VEGF）小片段干扰性RNA（siRNA）对小鼠视网膜新生血管的抑制作用。方法:C57BL/6J小鼠玻璃体腔注射pEGFP-N1-脂质体复合物,1d后视网膜铺片观察GFP的表达。选7d龄C57BL/6J小鼠117只,其中21只为正常组;另96只建立氧诱导的视网膜新生血管模型,并随机分为:模型对照组、空载体组和基因治疗组（HIF-1α组、VEGF组和共转染组）。于出舱前1d,采用脂质体介导的转染方法,向空载体组小鼠玻璃体腔注射空载体质粒;HIF-1α组注射HIF-1αsiRNA;VEGF组注射VEGF₁₆₅siRNA;共转染组注射HIF-1αsiRNA+VEGF₁₆₅siRNA。RT-PCR检测转染后重组质粒的表达;采用荧光造影视网膜铺片方法观察血管形态变化;组织切片观察并计算突破视网膜内界膜的血管内皮细胞核数;RT-PCR、免疫组化和Western blot检测视网膜HIF-1α和VEGF的表达。结果:RT-PCR检测发现小鼠视网膜中HIF-1αsiRNA和VEGF₁₆₅siRNA重组质粒有表达;pEGFP-N1经脂质体介导转染视网膜后1d即可见GFP表达;视网膜铺片可见基因治疗组较模型对照组和空载体组新生血管丛明显减少,荧光渗漏明显减轻,共转染组效果最明显;组织切片可见基因治疗组较其他三组突破视网膜内界膜的细胞核数量减少,差异均有统计学意义（P＜0.01）;视网膜HIF-1αmRNA及蛋白表达水平:模型对照组和空载体组较正常组上调,而HIF-1α组较模型对照组下调,抑制效率分别为57.4%和52.5%,差异有统计学意义（P＜0.01）。VEGF mRNA及蛋白表达水平:模型对照组和空载体组较正常组明显上调,而基因治疗组较模型对照组明显下调,差异均有统计学意义（P＜0.01）,共转染组下调最明显,抑制效率分别为85.6%和80.9%。结论:HIF-1α和VEGF₁₆₅siRNA均能有效抑制小鼠视网膜新生血管的形成,两者共转染抑制效果更明显。更多还原

【Abstract】 Chapter one:The inhibition of expression of HIF-1αand VEGF by small interfering RNA targeting HIF-1αand VEGF in the human vascular endothelial cellsObjective:To investigate the effect of small interfering RNA （siRNA）targeting hypoxia inducible factor-1α（HIF-1α）and vascular endothelial growth factor（VEGF）on expression of HIF-1αand VEGF in the human umbilical vein endothelial cells.Methods:HIF-1αsiRNA recombinant plasmid was constructed. Human umbilical vein endothelial cells were cultured in vitro and divided into normoxia group（20%）and hypoxia group（1%）.Hypoxia group was then divided into control hypoxia group,vector group,HIF-1αgroup, VEGF group and co-transfection group randomly.The cells were transiently transfected with vector plasmids（vector group）,HIF-1αsiRNA recombinant plasmid（HIF-1αgroup）,VEGF₁₆₅siRNA recombinant plasmid（VEGF group）,HIF-1αsiRNA and VEGF₁₆₅siRNA recombinant plasmids（co-transfection group）by the Lipofectamine 2000 （LF2000）method.No transfection were performed in the normoxia group and control,hypoxia group.The expression of HIF-1αsiRNA and VEGF₁₆₅siRNA recombinant plasmids were identified by reverse transcriptase- polymerase chain reaction（RT-PCR）.The expression of HIF-1αand VEGF were detected by RT-PCR and immunocytochemical methods.Results:The expression of HIF-1αsiRNA and VEGF₁₆₅siRNA recombinant plasmids were identified by RT-PCR after 24 hours of transfection to the cells.The exression of HIF-1αmRNA was observed in the normoxia group,whereas nearly no expression of HIF-1αprotein was observed.HIF-1αmRNA and protein levels of control hypoxia goup and vector group were increased compared to normoxia group.HIF-1αmRNA and protein levels of HIF-1αgroup decreased compared to control hypoxia group（P＜0.01）,mRNA level was reduced by 59.8%.The expression of VEGF mRNA and protein were faint in the normoxia group, but increased obviously after hypoxia（P＜0.01）.The expression of VEGF mRNA and protein in HIF-1αgroup,VEGF group and co-transfection group were decreased as compared with control hypoxia group（P＜0.01）, mRNA levels were reduced by 39.7%,68.1%and 82.3%,respectively. Co-transfection group showed the highest inhibitory effect.Conclusions:HIF-1αand VEGF₁₆₅siRNAs effectively inhibit the expression of HIF-1αand VEGF in the human umbilical vein endothelial cells.Chapter two:Inhibitory effect of small interfering RNA targeting HIF-1αand VEGF on retinal neovascularization in the mouseObjective:To evaluate the inhibitory effects of small interfering RNA（siRNA）targeting hypoxia inducible factor-1α（HIF-1α）and vascular endothelial growth factor（VEGF）on retinal neovascularization in the mouse.Methods:Liposome mediated pEGFP-N1 complex was injected into the vitreous of C57BL/6J mice.The expression of GFP was observed in retinal flat-mounts one day after injection.There were 21 seven-day-old C57BL/6J mice in the normal group and 96 mice which were divided into five groups randomly including control model group,vector group and gene therapy group（HIF-1αgroup,VEGF group and co-transfection group）were induced for retinal neovascularization by hypoxia.The mice received an intravitreous injection of vector plasmids（vector group）, HIF-1αsiRNA（HIF-1αgroup）,VEGF₁₆₅siRNA（VEGF group）,HIF-1αsiRNA and VEGF siRNA（co-transfection group）by the Lipofectamine 2000（LF2000）method 1 day before mice were moved out to room air from the oxygen chamber.The expression of HIF-1αsiRNA and VEGF₁₆₅ siRNA recombinant plasmids were identified by RT-PCR.Fluorescein angiography was used to assess the vascular pattern.The proliferative neovascular response was quantified by counting the nuclei of new vessels extending from retinas into the vitreous in cross-sections.HIF-1αand VEGF levels in retinas were measured by RT-PCR,Western blot and immunohistochemical methods.Results:The expression of HIF-1αsiRNA and VEGF₁₆₅siRNA recombinant plasmids were identified by RT-PCR.The GFP expression in retinal cells was observed one day after injection of liposome mediated pEGFP-N1 complex.Neovascular tufts and fluorescein leakage were decreased in gene therapy group especially co-transfection group compared to control model group and vector group.The neovascular nuclei were decreased in gene therapy group compared to the other three groups（P＜0.01）.The expression of HIF-1αmRNA and protein in retinas were increased in control model group and vector group as compared with normal group,while decreased 57.4%and 52.5%respectively in HIF-1αgroup as compared with control model group（P＜0.01）.The expression of VEGF mRNA and protein in retinas were increased significantly in control model group and vector group as compared with normal group,while decreased significantly in gene therapy group especially co-transfection group（decreased 85.6%and 80.9% respectively）as compared with control model group（P＜0.01）.Conclusions:HIF-1αand VEGF₁₆₅siRNAs can inhibit retinal neovascularization in the mouse effectively.Co-transfection of these two siRNAs shows the greatest inhibitory effect.更多还原