MOLECULAR INDUCTION OF CORNEAL ENDOTHELIAL PROLIFERATION

角膜内皮增殖的分子诱导

• 批准号: 6524981
• 负责人: NANCY C. JOYCE
• 金额: $ 34.8万
• 依托单位: SCHEPENS EYE RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6524981
• 关键词: aging; antisense nucleic acid; cell cycle; cell growth regulation; cell proliferation; corneal endothelium; electron microscopy; flow cytometry; gene expression; gene induction /repression; human tissue; immunocytochemistry; laboratory rabbit; molecular biology; organ culture; polymerase chain reaction; transcription factor; transfection; transmission electron microscopy; western blottings

The endothelium is the monolayer of cells that maintains corneal clarity. Corneal endothelial cell density decreases with age and cell loss can be accelerated by age-related diseases, and by ocular surgery or laser procedures. Maintenance of corneal clarity requires an intact endothelial monolayer, and transparency can be lost when cell density is reduced. Currently, visual loss caused by endothelial dysfunction can only be restored by corneal transplantation. Decreased cell density over time indicates that the rate of cell division does not keep pace with the rate of cell loss. The long-term goal of this project is to develop therapies to increase endothelial cell density. The proposed approach is to use molecular biology methods to transiently stimulate proliferation. Studies from this laboratory indicate that, in vivo, corneal endothelial cells are arrested in G1-phase (the part of the cell cycle that prepares cells for DNA synthesis) and are actively maintained in this non-replicative state. G1- phase arrest results from the activity of specific inhibitors that prevent induction of S-phase genes by the transcription factor, E2F. The hypothesis for these studies is that proliferation can be induced in corneal endothelial cells either by bypassing the point of G1-phase arrest and inducing the next downstream event in the cell cycle, or by decreasing the cellular concentration of G1-phase inhibitors. Two specific aims are proposed: 1) Stimulate proliferation and increase cell density in human corneal endothelium by overexpressing the transcription factor, E2F-2, and 2) Stimulate proliferation and increase cell density in human corneal endothelium by decreasing expression of the cell cycle inhibitor, p27kipl, using antisense technology. Both aims use methods that should induce a limited number of division cycles in a limited number of cells. This is based on the hypothesis that, to increase cell density to a level that will support endothelial function, only limited proliferation is necessary and desirable. For both aims, studies will be conducted in ex vivo human donor corneas. A rabbit model will also be used to permit evaluation at several levels of biological complexity, i.e., in tissue culture; in ex vivo corneal culture; and in treated ex vivo corneas transplanted back into a rabbit host. Immunocytochemistry (ICC), Western blots, and RT-PCR will detect alterations in E2F-2 and p27kip1 protein and mRNA expression. Proliferation will be detected by ICC, Western blot and RT-PCR for S- phase and cell cycle markers, and by flow cytometric analysis of DNA content. Cell density will be calculated by image analysis. Electron microscopy (TEM) and ICC for markers of plasma membrane polarity will evaluate endothelial morphology and ultrastructure following cDNA or antisense treatments. Endothelial function in treated, transplanted rabbit corneas will be tested in vivo by ultrasonic pachymetry measurements of corneal thickness. Apoptosis will be detected by annexin V assay and by gel assay of DNA fragments. TEM will detect multiple cell layers, providing evidence for cell transformation upon cDNA or antisense treatment.

内皮细胞是维持角膜透明度的单层细胞。角膜内皮细胞密度随着年龄的增长而降低，年龄相关性疾病以及眼部手术或激光手术可加速细胞损失。维持角膜透明度需要完整的内皮单层，当细胞密度降低时，透明度可能会丧失。目前，由内皮功能障碍引起的视力丧失只能通过角膜移植来恢复。随着时间的推移，细胞密度降低表明细胞分裂的速率与细胞损失的速率不同步。该项目的长期目标是开发增加内皮细胞密度的疗法。所提出的方法是使用分子生物学方法瞬时刺激增殖。该实验室的研究表明，在体内，角膜内皮细胞被阻滞在G1期（细胞周期中为DNA合成做准备的部分），并积极维持在这种非复制状态。G1期阻滞是由特异性抑制剂的活性引起的，该抑制剂阻止转录因子E2 F诱导S期基因。这些研究的假设是，可以通过绕过G1期阻滞点并诱导细胞周期中的下一个下游事件，或通过降低G1期抑制剂的细胞浓度来诱导角膜内皮细胞的增殖。提出了两个具体目标：1）通过过表达转录因子E2 F-2来刺激人角膜内皮细胞的增殖并增加细胞密度，以及2）通过使用反义技术降低细胞周期抑制剂p27 kipl的表达来刺激人角膜内皮细胞的增殖并增加细胞密度。这两个目标都使用了在有限数量的细胞中诱导有限数量的分裂周期的方法。这是基于这样的假设，即为了将细胞密度增加到将支持内皮功能的水平，仅有限的增殖是必要的和期望的。为了这两个目的，将在离体人供体角膜中进行研究。还将使用兔模型进行多个生物学复杂性水平的评价，即，在组织培养中;在离体角膜培养中;和在经处理的离体角膜移植回兔宿主中。免疫细胞化学（ICC），蛋白质印迹和RT-PCR将检测E2 F-2和p27 kip 1蛋白和mRNA表达的变化。将通过ICC、Western印迹和RT-PCR检测S期和细胞周期标志物，并通过流式细胞术分析DNA含量来检测增殖。将通过图像分析计算细胞密度。用于质膜极性标记物的电子显微镜（TEM）和ICC将评价cDNA或反义处理后的内皮形态和超微结构。将通过角膜厚度的超声测厚法测量在体内测试经处理的移植兔角膜中的内皮功能。将通过膜联蛋白V测定和DNA片段的凝胶测定来检测细胞凋亡。TEM将检测多个细胞层，为cDNA或反义处理后的细胞转化提供证据。