用于蛋白质组学分析的人视网膜及视网膜色素上皮-脉络膜解剖
Dissection of Human Retina and RPE-Choroid for Proteomic Analysis.
作者信息
Cabral Thiago, Toral Marcus A, Velez Gabriel, DiCarlo James E, Gore Anuradha M, Mahajan MaryAnn, Tsang Stephen H, Bassuk Alexander G, Mahajan Vinit B
机构信息
Barbara & Donald Jonas Stem Cell Laboratory, and Bernard & Shirlee Brown Glaucoma Laboratory, Department of Pathology & Cell Biology, Institute of Human Nutrition, College of Physicians and Surgeons, Columbia University; Edward S. Harkness Eye Institute, New York-Presbyterian Hospital; Department of Ophthalmology, Federal University of Sao Paulo (UNIFESP); Department of Ophthalmology, Federal University of EspÍrito Santo (UFES).
Omics Laboratory, Byers Eye Institute, Department of Ophthalmology, Stanford University; Medical Scientist Training Program, University of Iowa.
出版信息
J Vis Exp. 2017 Nov 12(129):56203. doi: 10.3791/56203.
The human retina is composed of the sensory neuroretina and the underlying retinal pigmented epithelium (RPE), which is firmly complexed to the vascular choroid layer. Different regions of the retina are anatomically and molecularly distinct, facilitating unique functions and demonstrating differential susceptibility to disease. Proteomic analysis of each of these regions and layers can provide vital insights into the molecular process of many diseases, including Age-Related Macular Degeneration (AMD), diabetes mellitus, and glaucoma. However, separation of retinal regions and layers is essential before quantitative proteomic analysis can be accomplished. Here, we describe a method for dissection and collection of the foveal, macular, and peripheral retinal regions and underlying RPE-choroid complex, involving regional punch biopsies and manual removal of tissue layers from a human eye.One-dimensional SDS-PAGE as well as downstream proteomic analysis, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), can be used to identify proteins in each dissected retinal layer, revealing molecular biomarkers for retinal disease.
人类视网膜由感觉神经视网膜和下方的视网膜色素上皮（RPE）组成，RPE与血管脉络膜层紧密结合。视网膜的不同区域在解剖学和分子层面上各不相同，具有独特的功能，并且对疾病的易感性也存在差异。对这些区域和层进行蛋白质组学分析，可以为包括年龄相关性黄斑变性（AMD）、糖尿病和青光眼在内的许多疾病的分子过程提供重要见解。然而，在进行定量蛋白质组学分析之前，分离视网膜区域和层是必不可少的。在此，我们描述了一种用于解剖和收集中央凹、黄斑和周边视网膜区域以及下方RPE-脉络膜复合体的方法，该方法涉及区域打孔活检以及从人眼中手动分离组织层。一维SDS-PAGE以及下游蛋白质组学分析，如液相色谱-串联质谱（LC-MS/MS），可用于鉴定每个解剖视网膜层中的蛋白质，揭示视网膜疾病的分子生物标志物。
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