CELL CYCLE OF THE CORNEAL ENDOTHELIUM

内皮是细胞的单层，位于细胞的后部 角膜，它保持角膜的透明度。在人类身上，角膜 内皮细胞密度随着年龄的增长而降低，这表明 有丝分裂替代细胞的速度与细胞损失率并不同步。 其他因素，如糖尿病、炎症、眼外伤或手术， 会导致细胞丢失，并可能导致内皮功能失调， 间质水肿，视力丧失。大疱性角膜病变由 内皮功能障碍目前在医学上是无法治疗和修复的 视力的恢复只能通过角膜移植来实现。我们的长- 学期目标是开发治疗方法以促进应激反应的愈合 角膜内皮。为了达到这个目标，我们必须发现角膜是如何 内皮修复是受调控的。正常情况下，有丝分裂和细胞运动 有助于单层修复；然而，人的角膜内皮有助于 损伤后不易分裂，因此修复主要通过细胞运动进行。 这种相对缺乏有丝分裂能力的现象并不普遍，因为角膜 兔等物种的内皮细胞很容易分裂。研究 在上一个赠款周期中，重点放在细胞运动的调节上。 拟议的研究将改变重点，集中于监管 角膜内皮细胞周期。这种方法更直接，而且可能 在实现长期目标方面更有帮助。工作假说 因为这些研究表明，尽管在人类中， 衰老的、不分裂的细胞随着年龄的增长而增加，在所有的年龄， 一群有丝分裂静止的G1期停滞细胞，它们是 能够刺激有丝分裂的。在人类和兔子身上，在体内 条件使内皮处于静止的、分化的状态 以保护其重要的生理功能。可能存在的因素 促成静止的因素包括接触抑制，相对较高 房水中转化生长因子-β(一种潜在的生长抑制因子)的浓度 体液，以及PGE2对自分泌有丝分裂的抑制。逆转增长停滞 在生长因子存在的情况下，如EGF，应该会促进有丝分裂 紧张的角膜内皮细胞。拟议的研究将使用CELL 生物、药理和分子生物学方法实现 具体目的如下：1)比较人和兔角膜 内皮细胞：活跃周期细胞和静止细胞的相对百分比 以及静止细胞在细胞周期内的相对位置 2)确定在胎儿早期发育期间，是否有 是人的角膜内皮从活跃的循环变为 与形成稳定的触点相关的静止状态 抑制和/或分化的单层，3)决定是否释放 避免接触抑制，特别是在EGF和/或 吲哚美辛可刺激角膜内皮细胞重新进入细胞 周期，4)确定转化生长因子-β是否抑制角膜内皮细胞 分裂，以及这种抑制是否可逆。这些研究应该 帮助确定人角膜的相对有丝分裂潜能 角膜内皮，研究体内角膜抑制的原因 内皮细胞有丝分裂并帮助发现逆转有丝分裂抑制的方法 以临床相关的方式。

The endothelium is the monolayer of cells, located at the posterior of the cornea, which maintains corneal transparency. In humans, corneal endothelial cell density decreases with age, suggesting that the rate of cell replacement by mitosis does not keep pace with the rate of cell loss. Other factors, such as diabetes, inflammation, ocular trauma or surgery, contribute to cell loss and can lead to endothelial decompensation, stromal edema, and loss of visual acuity. Bullous keratopathy caused by endothelial dysfunction is currently medically untreatable and restoration of vision can only be accomplished by corneal transplantation. Our long- term goal is to develop medical treatments to promote healing of stressed corneal endothelium. To reach this goal, we must discover how corneal endothelial repair is regulated. Normally, both mitosis and cell movement contribute to monolayer repair; however, human corneal endothelium does not readily divide upon injury, so repair occurs mainly by cell movement. This relative lack of mitotic capability is not universal, since corneal endothelial cells in species, such as rabbits, readily divide. Studies during the last grant cycle focussed on the regulation of cell movements. The proposed studies will change focus to concentrate on the regulation of the corneal endothelial cell cycle. This approach is more direct and may be more helpful in achieving the long-term goal. The working hypothesis for these studies is that, although in humans the relative number of senescent, non-dividing cells increases with age, there is, at all ages, a population of mitotically quiescent, G1-phase arrested cells which are capable of mitogenic stimulation. In both humans and rabbits, in vivo conditions maintain the endothelium in a quiescent, differentiated state to preserve its important physiologic functions. Factors which may contribute to quiescence include contact inhibition, the relatively high concentration of TGF-beta (a potential growth inhibitor) in the aqueous humor, and autocrine mitotic inhibition by PGE2. Reversal of growth arrest in the presence of growth factors, such as EGF, should promote mitosis in stressed corneal endothelium. The proposed studies will use cell biological, pharmacological and molecular biological methods to achieve the following Specific Aims: 1) Compare in human and rabbit corneal endothelium the relative percent of actively cycling and quiescent cells and the relative position within the cell cycle in which quiescent cells are arrested, 2) Determine whether, during early fetal development, there is a change in human corneal endothelium from an actively cycling to a quiescent state which correlates with the formation of a stable, contact inhibited and/or differentiated monolayer, 3) Determine whether release from contact inhibition, particularly in the presence of EGF and/or indomethacin can stimulate corneal endothelial cells to re-enter the cell cycle, and 4) Determine whether TGF-beta inhibits corneal endothelial cell division and whether such inhibition is reversible. These studies should help determine the relative mitotic potential of human corneal endothelium, investigate causes for in vivo inhibition of corneal endothelial mitosis and help discover means to reverse mitotic inhibition in a clinically relevant manner.