干扰RNA抑制小鼠视网膜HIF.docx

干扰RNA抑制小鼠视网膜HIF【摘要】目的：探讨缺氧诱导因子-1α特异性小片段干扰性RNA对小鼠视网膜HIF-1α蛋白质的抑制作用。方法：构建HIF-1αsiRNA重组质粒pSUPERsiHIF-1α。选择7d龄C57BL/6J小鼠24只，其中6只为正常组；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、空载体组和基因治疗组，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-1αsiRNA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-1αsiRNA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InhibitoryeffectofinterferingRNAtargetingHIF-1αproteininmiceretinaAbstractAIM:ToinvestigatetheinhibitoryeffectofinterferingRNAtargetingHIF-1αproteinintheretinaof:HIF-1αsiRNAwasconstructed(pSUPERsiHIF-1α).Therewere6seven-day-oldC57BL/6Jmiceinthenormalgroup(A),andanother18miceweredividedrandomlyintocontrolgroup(B),vectorgroup(C)andgenetherapygroup(D),with6miceineachgroup,whichwereinducedforretinalneovascularizationbyhypoxia.LiposomewithvectorplasmidandHIF-1αsiRNAwereinjectedintothevitreousinthevectorgroupandgenetherapygrouprespectivelyat1daybeforemiceweremovedouttoroomairfromthecabin.FITC-Dextranangiographywasusedtoobservethepatternoftheretinalvascular.TheexpressionofHIF-1αproteinamongeachgroupwasdetectedusingSP:Retinalflat-mountsafterFITC-DextranangiographyindicatedthatthevesselsofnormalmiceformedafinedradialbranchingpatterningroupA.Whilethelargevesselsweredistorted,thecapillarywasobstructed,neovascularclustersproliferatedandfluorescenceleakedintheperipheryoftheretinaingroupsBandC.AftertransfectionofHIF-1αsiRNAintotheretina,theretinalneovascularizationandnon-perfusiondistractioningroup)reducedandthestructureofretinaweremoreregularthanthoseingroupsBandC.ImmunohistochemicalstainingshowedthatHIF-1αexpressedmainlyintheretinalganglioncelllayeringroupsBandC,butnotingroupA.AftertransfectionofHIF-1αsiRNAintotheretina,theexpressionofHIF-1αwasdownregulatedcomparedwithgroup