Molecular Induction of Corneal Endothelial Proliferation

人角膜内皮细胞（HCEC）在细胞周期的G1期受到抑制。这可以防止细胞 分裂取代死细胞，并可能导致极低的细胞密度。我们的长期目标是 确定这种抑制的分子基础，并开发刺激增殖的方法，以增加 细胞密度HCEC在所有供体年龄都具有增殖能力;然而，存在内在的年龄依赖性。 相关的降低，其对血清的反应，这意味着随着供体年龄的增加抑制增加。的 我们研究的目标是确定这种与年龄相关的减少的抑制机制， 血清反应性一个令人兴奋的，新的方法将使用基于复制衰老的研究。 我们将检验以下假设：年龄相关的HCEC对血清反应能力的降低是由于 细胞周期蛋白依赖性激酶抑制剂p21 CIP 1（p21）的抑制作用增强。离体角膜和 在这些研究中将使用来自年轻（<30岁）和老年供体（> 50岁）的培养的HCEC。在目标1中，图像 Mcm 2表达的分析将允许计算每个年龄组中HCEC的百分比， 能够对血清做出反应。在Aim 2中，蛋白质印迹、实时PCR和迁移率改变 检测将测试是否存在与年龄相关的关键G1期调节因子活性的降低， p21抑制作用增强。目的3将测试培养细胞的p21 siRNA处理是否会促进增殖 从老年人捐赠者那里获得。目的4将测试p21 siRNA治疗是否会促进内皮细胞凋亡的增加。 来自老年供体的离体角膜中的细胞密度。目的3和4将使用细胞周期标志物染色， 直接细胞计数以记录增殖。在目标4中，图像分析将测试p21 siRNA处理是否将 促进密度<1500个细胞/mm 2的角膜中细胞密度的增加。如果我们的假设正确， 分子方法可以通过减少老年供体中HCEC的残余增殖能力， p21表达。

Human corneal endothelial cells (HCEC) are inhibited in G1-phase of the cell cycle. This prevents cell division from replacing dead cells and can result in critically low cell density. Our LONG-TERM GOALS are to identify the molecular basis for this inhibition and to develop methods to stimulate proliferation to increase cell density. HCEC at all donor ages possess proliferative capacity; however, there is an intrinsic, age- related decrease in their response to serum, implying increased inhibition with increasing donor age. The GOAL of our studies is to identify the inhibitory mechanisms responsible for this age-related decrease in serum responsiveness. An exciting, novel approach will be used based on studies of replicative senescence. We will test the HYPOTHESIS that the age-related decrease in ability of HCEC to respond to serum is due to increased inhibition by the cyclin-dependent kinase inhibitor, p21CIP1 (p21). Both ex vivo corneas and cultured HCEC from young (<30yo) and older donors (>50yo) will be used in these studies. In Aim 1, image analysis of Mcm2 expression will permit calculation of the percent of HCEC in each age-group that are competent to replicate in response to serum. In Aim 2, Western blots, real-time PCR, and mobility shift assays will test if there is an age-related decrease in the activity of key G1-phase regulators that suggests increased inhibition by p21. Aim 3 will test if p21 siRNA treatment of cultured cells will promote proliferation in HCEC from OLDER donors. Aim 4 will test if p21 siRNA treatment will promote an increase in endothelial cell density in ex vivo corneas from OLDER donors. Aims 3 and 4 will use staining for cell cycle markers and direct cell counts to document proliferation. In Aim 4, image analysis will test if p21 siRNA treatment will promote an increase in cell density in corneas with densities <1500 cells/mm2. If our hypothesis is correct, molecular approaches could tap the residual proliferative capacity of HCEC in older donors by decreasing p21 expression.