Molecular Induction of Corneal Endothelial Proliferation

描述：人角膜内皮细胞（HCEC）在细胞周期的g1期被抑制。这阻止了细胞分裂取代死细胞，并可能导致细胞密度极低。我们的长期目标是确定这种抑制的分子基础，并开发刺激增殖以增加细胞密度的方法。所有供体年龄的HCEC都具有增殖能力；然而，他们对血清的反应有一种内在的、与年龄相关的下降，这意味着随着供体年龄的增加，抑制作用增强。我们研究的目的是确定这种与年龄相关的血清反应性下降的抑制机制。基于对复制性衰老的研究，一种令人兴奋的、新颖的方法将被使用。我们将检验HCEC对血清反应能力的年龄相关性下降是由于细胞周期蛋白依赖性激酶抑制剂p21CIP1 （p21）的抑制增加的假设。这些研究将使用离体角膜和培养的年轻（<30岁）和老年供体（>50岁）的HCEC。在目标1中，Mcm2表达的图像分析将允许计算每个年龄组中能够响应血清复制的HCEC的百分比。在Aim 2中，Western blots、real-time PCR和迁移转移试验将测试关键g1期调节因子活性是否存在与年龄相关的下降，这表明p21的抑制作用增强。目的3将测试p21 siRNA处理培养细胞是否会促进老年供体HCEC的增殖。目的4将测试p21 siRNA治疗是否会促进老年供体离体角膜内皮细胞密度的增加。目标3和4将使用细胞周期标记染色和直接细胞计数来记录增殖。在Aim 4中，图像分析将测试p21 siRNA处理是否会促进密度<1500细胞/mm2的角膜细胞密度的增加。如果我们的假设是正确的，分子方法可以通过降低p21的表达来利用老年供体中HCEC的剩余增殖能力。

DESCRIPTION: 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.