脂质体介导缺氧诱导因子-1α特异性小干扰RNA重组质粒对小鼠视网膜新生血管的抑制作用  

Inhibition of liposome-mediated recombinant plasmid expressing small interference RNA targeting hypoxia- induced factor-1α on retinal neovascularization in mice

作　　者：熊宇[1] 夏晓波[1] 蒋剑[1] 宋伟涛[1] 

机构地区：[1]中南大学湘雅医院眼科,长沙410008

出　　处：《中华眼底病杂志》2013年第3期290-295,共6页Chinese Journal of Ocular Fundus Diseases

基　　金：国家自然科学基金（030471853）

摘　　要：【摘要】目的观察脂质体Lipofectamine“2000（LF2000）介导pSUPER为载体的缺氧诱导因子-1α（HIF-1α）特异性小干扰RNA重组质粒（pSUPERsi HIF-1α）对小鼠视网膜新生血管的抑制作用。方法构建重组质粒pSUPER-1α。将48只7日龄C57BL／6J小鼠分为正常组、空白对照组、空载体组和基因治疗组，每组12只。正常组小鼠在正常空气中饲养。空白对照组、空载体组和基因治疗组建立氧诱导的视网膜新生血管模型。后空白对照组小鼠不再作任何处理。于出舱前ld，空载体组小鼠玻璃体腔注射pSUPER和LF2000脂质体混合物1μl，基因治疗组小鼠玻璃体腔注射重组质粒pSUPER-1α和LF2000脂质体混合物1μl。采用荧光造影视网膜铺片观察各组小鼠视网膜血管形态变化；作病理切片，光学显微镜下计数各组突破视网膜内界膜的新生血管内皮细胞核数；免疫组织化学染色法检测各组小鼠视网膜中HIF-1α、血管内皮生长因子（VEGF）的蛋白表达；逆转录聚合酶联反应（RT—PCR）检测各组小鼠视网膜中HIF-1amRNA的表达。结果视网膜铺片荧光显微镜观察显示，正常组小鼠整个视网膜血管分布呈均匀网状；空白对照组、空载体组小鼠视网膜中周部可见大片无灌注区及新生血管丛，伴荧光渗漏；基因治疗组无灌注区及新生血管丛空白对照组、空载体组明显减少，视网膜血管网状结构基本正常。光学显微镜观察发现，空白对照组、空载体组突破视网膜内界膜的血管内皮细胞数较正常组明显增多，差异有统计学意义（F=5850．016，P〈O．05）；基因治疗组突破视网膜内界膜的血管内皮细胞数较空白对照组明显下降，差异有统计学意义（F=3012．469，P〈0．05）。免疫组织化学染色结果显示，HIF-1α蛋白阳性表达主要位于细胞核中，VEGF蛋白阳性表达主要位于细胞浆中。正常组小鼠视网膜中HIF-1α蛋白表达�Objective To observe the inhibition of Lipofectaminerm2000 （LF2000）-mediated pSUFER recombinant plasmid expressing small interference RNA targeting hypoxia-induced factor （HIF）-1α （pSUPER-1α） on retinal neovascularization in mice. Methods pSUPERSIHI-1α recombinant plasmid was created. Forty-eight （seven-day-old） C57BL/6J mice were randomly divided into a normal group, the control group, empty vector group and gene therapy group with 12 mice in each group. Mice in the normal group were kept in normal room air, while the other three groups retinal neovascularization was induced by bypoxia. The mice in control group were not treated. The mice in the vector group received intravitreousinjection of pSUPER and LF2000 （1 μl）, and the gene therapy group received pSUPER and LF2000 （1 μl） one day before being returned to normal room air. 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-luand vascular endothelial growth factor （VEGF） levels in retinas were measured by immune histochemical staining method and reverse transeriptase- polymerase chain reaction （RT-PCR）. Results Fluorescent angiography showed radial branching pattern vessels in the normal group and distorted large vessels, obstructed capillaries, many neovascular tuffs, fluorescence leakage in the peripheral retina in the control group and vector group. The gene therapy group demonstrated a significant reduction in neovascular tufts and fluorescence leakage compared with the control group and the vector group. The number of vascular cell nuclei extending breaking through the internal limiting membrane（lLM） of control group and vector group increased significantly compared with normal group （F= 5850. 016, P〈0.05）, while obviously decreasing in the gene therapy group compared with control group （F=3012. 469, P〈0.05）. Immunohistochemicat sta

关 键 词：视网膜新生血管化 药物疗法 缺氧诱导因子-1α亚基 脂质体 基因转移技术 动 物实验 

分 类 号：R77[医药卫生—眼科]