CELL BIOLOGY OF CORNEAL ENDOTHELIAL DYSFUNCTION

为了了解内皮细胞背后的细胞因素 人类角膜的功能障碍，我们建议首先来看看一些 角膜内皮再生能力的细胞参数 细胞。我们将在伤口愈合过程中加强内皮细胞的再生 使用两个动物系统，其中一个具有很高的再生能力 (兔子)，和一个再生能力有限的(猫)。我们的 方法将重点放在膜相关分子的变化和 用凝集素标记血管内皮细胞的屏障功能 冷冻断裂(FF)技术作为细胞膜变化的探针， 和电子显微镜(EM)Na+/K+-ATPase的定位和示踪 渗透性作为屏障功能的探头。其次，我们将比较 在人体组织中的相同参数，不仅限于它们的 再生能力，但已经变得功能失调，使用 无晶状体眼患者移植手术中内皮细胞的获取 大泡性角膜病变、人工晶状体大泡性角膜病变和Fuchs病 内皮营养不良。作为对照，未受伤的眼睛将被 与受伤的眼睛相比，人类的内皮功能障碍将是 与圆锥角膜移植眼的正常内皮细胞相比 年龄匹配的眼库眼睛。目标是首先通过以下方式进行分析和量化 EM兔和兔的凝集素-铁蛋白结合位点数 猫再生和人类功能障碍的角膜内皮细胞。 其次，用Ff和Ff细胞化学方法分析膜内的变化 在再生的兔和猫内皮细胞和人中发生 内皮细胞功能失调。我们将注意分析 细胞间连接，膜内颗粒分布，以及 这些复制品上的糖偶联物的细胞化学鉴定。第三， 高效毛细管电泳法分析菲立平-甾醇复合体在兔和大鼠体内的分布 再生期和人的CAT内皮膜 膜功能失调。已知膜中的胆固醇会影响 胞饮活动率也可能影响通透性和 内皮细胞的屏障功能。第四，研究细胞内 细胞外示踪剂(辣根过氧化物酶)的摄取和转运 跨兔和猫角膜内皮细胞在再生和 横跨人类功能失调的内皮细胞。最后，对Na+/K+进行本地化 兔和猫再生内皮细胞对ATPase活性的依赖性 在人类功能障碍的内皮细胞中，使用EM细胞化学。

In order to understand the cellular factors underlying endothelial dysfunction in the human cornea, we propose first to look at some of the cellular parameters of the regenerative ability of corneal endothelial cells. We will stury regenerating endothelial cells during wound healing using two animal systems, one which has a high regenerative capacity (rabbit), and one which has a limited regenerative capacity (cat). Our approach will focus on changes in membrane-associated molecules and the barrier function of the endothelium, using lectin-labeling and freeze-fracture (FF) techniques as cellular probes for membrane changes, and electron microscopic (EM) Na+/K+-ATPase localization and tracer permeability as probes for barrier function. Secondly, we will compare the same parameters in human tissues, which are not only limited in their regenerative capacity, but which have become dysfunctional, using endothelium obtained during transplant surgery from patients with aphakic bullous keratopathy, pseudophakic bullous keratopathy, and Fuchs' endothelial dystrophy. For control comparison, unwounded eyes will be compared with wounded eyes and human dysfunctional endothelium will be compared with normal endothelium from keratoconus transplant eyes or age-matched eye bank eyes. The aims are to first analyze and quantitate by EM the number of lectin-ferritin binding sites on rabbit and cat-regenerating and human-dysfunctional corneal endothelial cells. Secondly, to analyze by FF and FF cytochemistry, the intramembrane changes occurring in regenerating rabbit and cat endothelial cells and in human dysfunctional endothelial cells. Attention will be given to analyzing intercellular junctions, intramembrane particle distribution, and cytochemical identification of glycoconjugates on these replicas. Thirdly, to analyze by FF the distribution of filipin-sterol complexes in rabbit and cat endothelial membranes during the regenerative phase and in human dysfunctional membranes. Cholesterol in membranes is known to affect the rates of pinocytotic activity which may also affect permeability and barrier functions of the endothelium. Fourthly, to study intracellular uptake and transport of an extracellular tracer (horseradish peroxidase) across rabbit and cat corneal endothelial cells during regeneration and across human dysfunctional endothelium. Lastly, to localize Na+/K+ dependent ATPase activity of regenerating rabbit and cat endothelial cells and in human dysfunctional endothelium, using EM cytochemistry.