干扰RNA抑制小鼠视网膜HIF-1α蛋白质表达.docVIP

干扰RNA抑制小鼠视网膜HIF-1α蛋白质表达 　 作者：熊宇　夏晓波　许惠卓　蒋剑　宋伟涛　刘双珍　许雪亮 【摘要】 　　目的：探讨缺氧诱导因子-1α（HIF-1α）特异性小片段干扰性RNA（siRNA）对小鼠视网膜HIF-1α蛋白质的抑制作用。方法：构建HIF-1α siRNA重组质粒pSUPERsiHIF-1α。选择7d龄C57BL/6J小鼠24只，其中6只为正常组（A）；另18只建立缺氧诱导的视网膜新生血管模型，并随机分为：对照组（B）、空载体组（C组）和基因治疗组（D组），每组6只。于出舱前1d，向空载体组小鼠玻璃体腔注射pSUPER空载体质粒；基因治疗组注射HIF-1α siRNA 重组质粒pSUPERsiHIF-1α。采用FITC-Dextran荧光造影视网膜铺片观察4组小鼠视网膜血管形态变化； SP免疫组织化学检测各组间HIF-1α的表达差异。 结果：荧光造影视网膜铺片显示，A组整个视网膜血管分布呈均匀网状；B、C组视网膜中周部可见大片无灌注区，见新生血管丛，伴荧光渗漏；D组无灌注区较B，C组明显减少，周边部毛细血管网基本正常，新生血管丛明显减少。免疫组织化学染色显示，A组HIF-1α蛋白表达阴性；B，C组在神经节细胞层呈阳性表达； D组在神经节细胞层呈弱阳性表达，较B，C组明显减少。 结论：采用玻璃体腔注射，通过脂质体介导HIF-1α siRNA重组质粒pSUPERsiHIF-1α转染视网膜，有效地抑制了缺氧诱导的小鼠视网膜新生血管模型视网膜中HIF-1α蛋白质的表达及视网膜新生血管的形成。 【关键词】 小干扰RNA　缺氧诱导因子-1α　视网膜新生血管　基因治疗 　　 Inhibitory effect of interfering RNA targeting HIF-1α protein in mice retina Abstract AIM: To investigate the inhibitory effect of interfering RNA targeting HIF-1α protein in the retina of mice.METHODS: HIF-1α siRNA was constructed (pSUPERsiHIF-1α). There were 6 seven-day-old C57BL/6J mice in the normal group (A), and another 18 mice were divided randomly into control group (B), vector group (C) and gene therapy group (D), with 6 mice in each group, which were induced for retinal neovascularization by hypoxia. Liposome with vector plasmid and HIF-1α siRNA were injected into the vitreous in the vector group and gene therapy group respectively at 1 day before mice were moved out to room air from the cabin. FITC-Dextran angiography was used to observe the pattern of the retinal vascular. The expression of HIF-1α protein among each group was detected using SP immunohistochemistry.RESULTS: Retinal flat-mounts after FITC-Dextran angiography indicated that the vessels of normal mice formed a fined radial branching pattern in group A. While the large vessels were distorted, the capillary was obstructed, neovascular clusters proliferated and fluorescence leaked in the periphery of the retina in groups B and C. After transfection of HIF-1α siRNA into the retina, the retinal neovascu