SOD2 knockdown mouse model of early AMD
SOD2 knockdown mouse model of early AMD
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DOI：
10.1167/iovs.07-0432
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发表时间：
2007-10-01
影响因子：
4.4
通讯作者：
Lewin, Alfred S.
中科院分区：
文献类型：
--
作者：
Justilien, Verline;Pang, Ji-Jing;Lewin, Alfred S.
目的。为了验证视网膜色素上皮（RPE）氧化损伤可能导致视网膜损伤的假设，类似于与年龄相关的黄斑变性（AMD）的早期阶段。在RPE-J细胞中表达了一种靶向保护酶锰超氧化物歧化酶（MnSOD）的核酶，并在成年C57BL/6小鼠视网膜下注射了表达该核酶基因的腺相关病毒（AAV）。利用免疫印迹分析和LC - MS/MS鉴定蛋白质和硝化位点，检测RPE/脉络络合物中SOD2蛋白水平和氧化损伤蛋白标志物。从视网膜组织中提取脂质，分析双视黄醇类化合物A2E和isoa2e。用全视场视网膜电图（ERG）观察小鼠的光反应。光镜和电镜观察视网膜的细胞学变化。Rz432处理RPE-J细胞导致MnSOD mRNA和蛋白水平降低，超氧阴离子水平升高，细胞凋亡。当通过AAV递送时，Rz432降低了MnSOD蛋白，增加了小鼠rpe -脉络中氧化损伤的标志物，包括硝化和羧乙基吡咯修饰的蛋白。核酶的递送导致视网膜电反应的逐渐丧失、空泡化、RPE变性、布鲁氏膜增厚、光感受器内外段的缩短和解体。细胞凋亡导致光感受器外核层逐渐变薄。与AMD患者的眼睛相似，核酶治疗的眼睛表现出增加的自身荧光和升高的A2E和isoa2e水平，这是脂褐素的主要双维甲酸色素。这些结果支持了RPE的氧化损伤可能在AMD的一些关键特征中起作用的假设。
PURPOSE. To test the hypothesis that oxidative injury to the retinal pigment epithelium (RPE) may lead to retinal damage similar to that associated with the early stages of age-related macular degeneration (AMD).METHODS. A ribozyme that targets the protective enzyme manganese superoxide dismutase ( MnSOD) was expressed in RPE-J cells, and adeno-associated virus (AAV) expressing the ribozyme gene was injected beneath the retinas of adult C57BL/6 mice. The RPE/choroid complex was examined for SOD2 protein levels and protein markers of oxidative damage using immunoblot analysis and LC MS/MS-identification of proteins and nitration sites. Lipids were extracted from retinal tissue and analyzed for the bis-retinoid compounds A2E and iso-A2E. The mice were analyzed by full-field electroretinography ( ERG) for light response. Light and electron microscopy were used to measure cytological changes in the retinas.RESULTS. The treatment of RPE-J cells with Rz432 resulted in decreased MnSOD mRNA and protein as well as increased levels of superoxide anion and apoptotic cell death. When delivered by AAV, Rz432 reduced MnSOD protein and increased markers of oxidative damage, including nitrated and carboxyethylpyrrole-modified proteins in the RPE-choroid of mice. Ribozyme delivery caused a progressive loss of electroretinograph response, vacuolization, degeneration of the RPE, thickening of Bruch's membrane, and shortening and disorganization of the photoreceptor outer and inner segments. Progressive thinning of the photoreceptor outer nuclear layer resulted from apoptotic cell death. Similar to the eyes of patients with AMD, ribozyme-treated eyes exhibited increased autofluorescence and elevated levels of A2E and iso-A2E, major bis-retinoid pigments of lipofuscin.CONCLUSIONS. These results support the hypothesis that oxidative damage to the RPE may play a role in some of the key features of AMD.