p27Kip1 and RPE Cell Cycle

描述（由申请人提供）：视网膜色素上皮（RPE）的异常生长或更新与惊人数量的眼部病症（即，增殖性玻璃体视网膜病变（PVR）、年龄相关性黄斑变性、发育性错构瘤、先天性RPE肥大），占显著的健康成本。了解建立和维持适当的上皮细胞数量和大小的基本机制对于找到减轻疾病后果和促进其修复的方法至关重要。这项研究的目的是确定在RPE原位发育过程中，通过细胞增殖和细胞大小扩大来调节生长的机制。虽然许多外源性影响可以调节细胞增殖，但它们最终是通过影响细胞分裂周期的机制来实现的。因此，所提出的研究集中在RPE细胞在体内上皮层分化期间退出细胞周期时的机制上。实验将使用具有细胞周期调节蛋白p27Kip1编码基因的靶向破坏的突变小鼠，其中存在RPE单层的异常扩增。总的假设是，p27 Kip1的丢失导致上皮细胞的数量或密度增加，这是由于细胞增殖速率的增强或分化前细胞周期退出时间的延迟。这一假设将使用免疫组织化学技术进行测试，以确定细胞分裂的幅度，以及细胞死亡，在不同的发育年龄。将通过共聚焦显微镜获得的图像的形态测定分析来检查RPE层增厚是由于细胞大小增加的替代假设。

DESCRIPTION (provided by applicant): Abnormal growth or turnover of the retinal pigment epithelium (RPE) is associated with an alarming number of ocular disorders (i.e., proliferative vitreo-retinopathy (PVR), age-related macular degeneration, developmental hamartomas, congenital hypertrophy of the RPE), accounting for significant health costs. An understanding of the basic mechanisms for establishing and maintaining appropriate epithelial cell number and size is crucial to finding ways to alleviate disease consequences and to facilitate their repair. The aim of the proposed research is to define the mechanisms that regulate growth, through cell multiplication and enlargement of cell size, during RPE development in situ. While many exogenous influences can regulate cell proliferation, they do so by ultimately impinging on machinery of the cell division cycle. Consequently, the proposed studies focus on mechanisms operating in the RPE cells as they withdraw from the cell cycle during differentiation of the epithelial layer in vivo. Experiments will make use of a mutant mouse with targeted disruption of the gene coding for the cell cycle regulatory protein p27Kip1, in which there is an abnormal expansion of the RPE rnonolayer. The overall hypothesis is that loss of p27Kip1 leads to an increase in the number or density of epithelial cells as a result of an enhancement in the rate of cell proliferation, or of a delay in the timing of cell cycle exit prior to differentiation. This hypothesis will be tested using immunohistochemical techniques to determine the magnitude of cell division, as well as cell death, at various developmental ages. The alternative hypothesis that the thickening of the RPE layer is due to an increase in cell size will be examined by morphometric analysis of images obtained by confocal microscopy.