BLOOD-RETINAL BARRIER GLUT1 IN DIABETIC RETINOPATHY

本申请中概述的项目的总体目标 是提供以研究为中心的基于导师的培训计划 糖尿病患者葡萄糖转运蛋白GLUT1基因表达的变化 视网膜病变(DR)。这个项目的基本原理是基于临床 长期糖尿病慢性高血糖的观察 与视网膜特有的病理改变有关 毛细血管构成血-视网膜内屏障(BRB)。vt.在.上 分子水平上，才能启动生化和 组织病理学改变导致DR，葡萄糖必须获得 内侧毛细血管内皮细胞的胞内室 BRB。它只能通过GLUT1的运输来做到这一点。因此，变化在 Glut1在BRB内部的表达可能对 细胞内葡萄糖浓度及其对后续发育的影响 的病理性微血管病变最近的一项初步研究 由申请人展示了一种局部的病理性上调 长期糖尿病患者BRB内侧的GLUT1免疫反应。这个 拟议的研究将在4个一般领域调查这一现象。 首先，定量免疫金电子显微镜和原位 将在死后的人视网膜上进行GLUT1的杂交 标本扩大对GLUT1蛋白和mRNA变化的观察 早期DR的表达第二，选择性血管过度表达 牛视网膜内皮细胞(BRCEC)培养中的内皮GLUT1 并在转基因小鼠模型中进行实验，以确定 GLUT1表达增加导致分子和组织病理学 与早期博士发现的变化类似第三，葡萄糖的影响 与GLUT1基因上DR发生相关的生长因子 将在BRCEC培养模型中研究表达，第四， 周细胞-内皮细胞相互作用对GLUT1基因的可能影响 表达将在细胞培养中进行研究。

The overall objective of the project outlined in the present application is to provide a mentor-based training program centered around the study of changes in GLUT1 glucose transporter gene expression in diabetic retinopathy (DR). The rationale for this project is based on the clinical observation that the chronic hyperglycemia of long-standing diabetes is associated with characteristic pathological changes in the retinal capillaries which comprise the inner blood-retinal barrier (BRB). On a molecular level, in order to initiate the biochemical and histopathological changes leading to DR, glucose must gain access to the intracellular compartment of the capillary endothelial cells of the inner BRB. It can only do so via transport by GLUT1. Therefore, changes in GLUT1 expression in the inner BRB may have a profound impact on the intracellular concentration of glucose and on the subsequent development of the pathological microvascular changes of DR. A recent pilot study by the applicant demonstrated a localized, pathological upregulation of immunoreactive GLUT1 on the inner BRB in long-standing diabetes. The proposed studies will investigate this phenomenon in 4 general areas. First, quantitative immunogold electron microscopy and in situ hybridization for GLUT1 will be performed on postmortem human retina specimens to extend observations regarding changes GLUT1 protein and mRNA expression in early DR. Second, selective overexpression of vascular endothelial GLUT1 in bovine retinal endothelial cells (BRCEC) in culture and in a transgenic mouse model will be undertaken to determine if increased expression of GLUT1 results in molecular and histopathological changes similar to those found in early DR. Third, the effects of glucose and growth factors associated with the development of DR on GLUT1 gene expression will be investigated in BRCEC culture models, and fourth, possible effects of pericyte-endothelial cell interactions on GLUT1 gene expression will be studied in cell culture.