MOLECULAR INDUCTION OF CORNEAL ENDOTHELIAL PROLIFERATION

内皮细胞是维持角膜透明的单层细胞。角膜内皮细胞密度随着年龄的增长而减少，与年龄相关的疾病以及眼科手术或激光手术都会加速细胞的丧失。维持角膜透明度需要完整的内皮单层，当细胞密度降低时，透明度可能会丢失。目前，由于内皮功能障碍导致的视力丧失只能通过角膜移植来恢复。细胞密度随着时间的推移而下降，表明细胞分裂的速度与细胞丢失的速度并不同步。该项目的长期目标是开发增加内皮细胞密度的治疗方法。建议的方法是使用分子生物学方法来瞬时刺激增殖。该实验室的研究表明，在体内，角膜内皮细胞被阻止在G1期(细胞周期中为DNA合成做准备的部分)，并活跃地维持在这种非复制状态。G1期停滞是由于特定的抑制物的活性导致的，这种抑制物可以阻止转录因子E2F诱导S期基因的表达。这些研究的假设是，通过绕过G1期停滞点并诱导细胞周期中的下一个下游事件，或者通过降低G1期抑制物的细胞浓度，可以诱导角膜内皮细胞的增殖。通过转录因子E2F-2的过表达来刺激人角膜内皮细胞的增殖和增加细胞密度，以及通过反义技术降低细胞周期抑制因子p27kipl的表达来刺激人角膜内皮细胞的增殖和增加细胞密度。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。这是基于这样一个假设：为了将细胞密度提高到支持内皮功能的水平，只有有限的增殖是必要和可取的。为了达到这两个目标，将在体外人类供体角膜中进行研究。兔模型也将被用来在几个级别的生物学复杂性上进行评估，即组织培养、体外角膜培养和经处理的体外角膜移植回兔宿主。免疫细胞化学(ICC)、Western blots和RT-PCR将检测E2F-2和p27kip1蛋白和mRNA表达的变化。采用免疫细胞化学、Western印迹和RT-PCR法检测S期细胞和细胞周期标志物的增殖情况，并用流式细胞仪检测细胞的DNA含量。细胞密度将通过图像分析来计算。用电子显微镜和ICC作为质膜极性的标记物，将评估经cDNAs或反义基因处理后的内皮细胞的形态和超微结构。治疗后的移植兔角膜的内皮功能将通过测量角膜厚度的超声测厚仪进行体内测试。用Annexin V法和DNA片段凝胶分析法检测细胞凋亡。透射电子显微镜将检测到多个细胞层，为细胞在cDNA或反义治疗中的转化提供证据。

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.