BLOOD-RETINAL BARRIER GLUT1 IN DIABETIC RETINOPATHY

本申请中概述的项目的总体目标 是提供一个以研究为中心的基于导师的培训计划， 糖尿病患者GLUT 1葡萄糖转运蛋白基因表达的变化 视网膜病变（DR）。本项目的基本原理是基于临床 观察到长期糖尿病的慢性高血糖是 与视网膜的特征性病理变化相关 毛细血管，包括内部血视网膜屏障（BRB）。上 分子水平，以启动生物化学和 组织病理学变化导致DR，葡萄糖必须获得进入 内毛细血管内皮细胞的细胞内区室 BRB。它只能通过GLUT 1传输来实现。因此， GLUT 1在BRB内的表达可能对BRB的形成有深远的影响。 细胞内葡萄糖浓度和随后的发展 DR的病理微血管变化。 申请人的研究表明， 免疫反应性GLUT 1在长期糖尿病的内部BRB。的 拟议的研究将在4个一般领域调查这一现象。 首先，定量免疫金电镜和原位 将在死后人视网膜上进行GLUT 1的杂交 标本，以扩展关于GLUT 1蛋白和mRNA变化的观察 第二，选择性过度表达血管内皮细胞， 培养的牛视网膜内皮细胞（BRCEC）中的内皮GLUT 1 并在转基因小鼠模型中进行，以确定 GLUT 1表达增加导致分子和组织病理学变化 变化与早期DR中发现的变化相似。第三，葡萄糖的影响 GLUT 1基因与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GLUT 1基因的可能影响 将在细胞培养中研究表达。

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.