机构地区:[1]中南大学湘雅医院眼科,长沙410008 [2]美国Texas大学MD Anderson癌症研究中心
出 处:《中华眼科杂志》2008年第10期921-928,共8页Chinese Journal of Ophthalmology
基 金:国家自然科学基金资助项目(30471853)
摘 要: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。方法本研究采用随机对照方法。构建HIF-1α siRNA重组质粒。C57BL/6J小鼠玻璃体腔注射增强型绿色荧光蛋白(EGFP)表达质粒pEGFP-N1,1d后视网膜铺片观察绿色荧光蛋白(GFP)的表达。选7天龄C57BL/6J小鼠90只,17只为正常组,73只建立氧诱导的视网膜新生血管模型,分为模型对照组、空载体组和基因治疗组(HIF1α siRNA组、VEGF siRNA组及共转染组)。于出氧舱前1d,向空载体组小鼠玻璃体腔注射空载体质粒;HIF—1α siRNA组注射HIF—1α siRNA,VEGF siRNA组注射VEGF165 siRNA,共转染组注射HIF1α siRNA+VEGF165 siRNA。采用荧光造影视网膜铺片方法观察血管形态变化;制作组织切片计算突破视网膜内界膜的血管内皮细胞核数;采用逆转录聚合酶链反应和Western blot检测视网膜HIF—1α和VEGF的表达。采用单因素方差分析和组间最小显著差法进行统计学分析。结果pEGFP-N1经脂质体LF2000介导转染视网膜细胞后1d即可见GFP表达。基因治疗组较模型对照组新生血管丛明显减少,荧光渗漏明显减轻,其中共转染组效果最明显。基因治疗组[HIF-1α siRNA组为(27.73±2.33)个,VEGF siRNA组为(15.43±3.23)个,共转染组为(8.70±2.88)个]较其他3组突破视网膜内界膜的细胞核数量减少,差异均具有统计学意义(F=3016.527,P〈0.01)。视网膜HIF-1α mRNA及蛋白表达水平:模型对照组(1.08±0.06,0.383±0.009)和空载体组(1.09±0.05,0.386±0.010)较正常组(0.81±0.07,0.035±0.003)上调,而HIF-1α siRNA组(0.46±0.06,0.182±0.008)较模型对照组下调,抑制效率分别为57.4%和52.5%,差异均有统计学意义(F=139.804,2686.001;P〈0.01)。VEGF mRNA及蛋白�Objective To evaluate the inhibitory effect of small interfering RNA (siRNA) on the expressions of hypoxia inducible factor-let (HIF-1α) and vascular endothelial growth factor (VEGF) in retinal neovascularization in the mouse. Methods HIF-1α siRNA recombinant plasmid was constructed. Liposome mediated the expressive plasmid of enhanced green fluorescent protein (EGFP) 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. Randomized controlled trial was performed. There were totally ninety (seven-day-old) C57BL/6J mice in which seventeen mice were chosen as normal group and seventy-three mice were divided into five groups randomly including control model group, vector group and gene therapy group ( HIF-1α siRNA group, VEGF siRNA group and co-transfection group), in which retinal neovasculafization was induced by hypoxia. Liposome with vector plasmid, HIF-1α siRNA and VEGF165 siRNA were injected into the vitreous in the vector group, HIF-1α siRNA group and VEGF siRNA group respectively one day before mice were moved out to room air from the cabin. Liposome with HIF-1αsiRNA and VEGF165 siRNA was injected in the co-transfection group at the same time point. Fluorescent angiography was used to assess the vascular pattern. The proliferative neovascular response was quantified by counting the nuclei of new vessels extending from the retina into the vitreous in cross-sections. HIF-1α and VEGF levels in retinas were measured by reverse transeriptase-polymerase chain reaction and Western blot. Significant differences between groups were evaluated by one-way analysis of variance, followed by a least-significant difference analysis. Results 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 in co-transfection group compared to the control model group. T
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