AAV介导HO-1基因转染对视网膜色素变性大鼠视网膜的保护作用  

Protective effect of adeno-associated virus vector mediated heme oxygenase-1 overexpression on retinitis pigmentosa in rats

作　　者：梁郁萍 陈蔚琪 洪玉[1] 高秀云[1] 郭茂生[1] Liang Yuping;Chen Weiqi;Hong Yu;Gao Xiuyun;Guo Maosheng(Department of Ophthalmology,The Second Affiliated Hospital of Fujian Medical University,Quanzhou 362000,China)

机构地区：[1]福建医科大学附属第二医院眼科,泉州362000

出　　处：《中华实验眼科杂志》2021年第8期693-699,共7页Chinese Journal Of Experimental Ophthalmology

基　　金：福建省自然科学基金项目(2017J01274);福建省卫生系统中青年骨干人才培养项目(2015-ZQN-JC-26);泉州市科技计划重点项目(2016Z053)。

摘　　要：目的探讨腺相关病毒(AAV)载体介导血红素加氧酶1(HO-1)基因过表达对视网膜色素变性(RP)模型大鼠的保护作用。方法选取健康雄性SD大鼠80只,体质量200~250 g,采用完全随机分组设计,将大鼠分为空白对照组、RP模型组、AAV-GFP组和AAV-HO-1-GFP组,每组20只。RP模型组、AAV-GFP组和AAV-HO-1-GFP组以剂量50 mg/kg的质量分数1%碘酸钠溶液进行尾静脉注射,制备碘酸钠诱导大鼠RP模型,再分别予以0.9%氯化钠溶液、AAV-GFP病毒、AAV-HO-1-GFP病毒视网膜下腔注射;空白对照组大鼠尾静脉仅注射等量0.9%氯化钠溶液。于造模后第14天摘出大鼠眼球。采用苏木精-伊红染色法检测各组大鼠视网膜结构及厚度;采用TUNEL法检测各组视网膜组织细胞凋亡情况;采用Western blot法测定各组大鼠视网膜组织中含半胱氨酸的天冬氨酸蛋白水解酶-3(caspase 3)、B淋巴细胞瘤-2(bcl-2)、HO-1蛋白的表达水平。结果苏木精-伊红染色结果显示,空白对照组大鼠视网膜结构正常,RP模型组和AAV-GFP组大鼠视网膜结构破坏明显,外核层呈波浪状改变,厚度变薄,AAV-HO-1-GFP组大鼠视网膜结构轻度破坏,外核层厚度轻度变薄。与空白对照组比较,RP模型组和AAV-GFP组大鼠视网膜外核层厚度明显变薄,差异均有统计学意义(均P<0.05)。RP模型组、AAV-GFP组和AAV-HO-1-GFP组HO-1蛋白相对表达量分别为0.76±0.21、0.76±0.16、0.92±0.05,明显高于空白对照组的0.48±0.25,差异均有统计学意义(均P<0.05);AAV-HO-1-GFP组HO-1蛋白相对表达量高于RP模型组和AAV-GFP组,差异均有统计学意义(均P<0.05)。各组细胞凋亡率、bcl-2和caspase 3蛋白相对表达量总体比较,差异均有统计学意义(F=1596.333、1043.806、364.331,均P<0.01);与AAV-HO-1-GFP组比较,RP模型组和AAV-GFP组细胞凋亡率和caspase 3蛋白相对表达量增加,bcl-2蛋白相对表达量减少,差异均有统计学意义(均P<0.05)。结论AAV介导HO-1基因过表达对RP模型�Objective To investigate the protective effect of adeno-associated virus(AAV)vector-mediated heme oxygenase-1(HO-1)gene overexpression on retinitis pigmentosa(RP)models in rats.Methods Eighty healthy male SD rats were selected and randomized into the blank control group,RP model group,AAV-GFP group and AAV-HO-1-GFP group,with 20 rats in each group.The RP model was established via tail vein injection of 1%sodium iodate solution at the dose of 50 mg/kg.Rats in the RP model group,AAV-GFP group and AAV-HO-1-GFP group were subretinally injected with normal saline,AAV-GFP virus and AAV-HO-1-GFP virus according to grouping.Rats in the blank control group were administrated with subretinal injection of equal volume of normal saline.The eyeballs of rats were enucleated on 14th day after molding.The retinal structure and thickness were detected by hematoxylin-eosin staining;apoptosis in retinal tissue was detected by TUNEL assay;the expression levels of cysteinyl aspartate specific proteinase 3(caspase 3),B-lymphoma-2(bcl-2)and HO-1 protein in rats retina was identified by Western blot.The use and care of the animals complied with the Statement of the Association for Research in Vision and Ophthalmology(ARVO).The study protocol was approved by an Ethics Committee of The Second Affiliated Hospital of Fujian Medical University(No.202188).Results Hematoxylin-eosin staining results showed normal retinal structure in the blank control group,obvious retinal structure damage with thinned and wavy outer nuclear layer in the RP model group and AAV-GFP group,and mild retinal structure damage with slightly thinned outer nuclear layer in the AAV-HO-1-GFP group.Compared with the blank control group,the outer nuclear layer was significantly thinner in the RP model group and AAV-GFP group(both at P<0.05).Relative expression level of HO-1 protein in the RP model group,AAV-GFP group,and AAV-HO-1-GFP group was 0.76±0.21,0.76±0.16,0.92±0.05,respectively,which were all significantly higher than 0.48±0.25 in the blank control group(all at

关 键 词：血红素加氧酶-1 视网膜色素变性 腺相关病毒 凋亡 

分 类 号：R774.1[医药卫生—眼科]