Proteases in the Cornea

外行描述：角膜的伤口愈合尚不完全清楚；然而每年都有成千上万的人 选择矫正的光屈光手术。确定参与伤口愈合的机制将导致 为那些不能正确愈合的患者开发治疗方法。在这项提案中，凝血酶，一种蛋白酶 产生纤维蛋白并调节细胞过程的物质，将被研究。科学描述：这一目标 应用于检验凝血酶通过裂解凝血酶参与角膜伤口愈合的假说 蛋白水解酶激活受体和信号通路的启动。我们的初步数据显示， 人角膜中凝血酶原转化为凝血酶和蛋白水解酶激活受体所需的基因 以及凝血酶改变角膜基质细胞基因表达和细胞分裂的能力。凝血酶 抑制剂水飞蓟素抑制角膜上皮伤口愈合。这项建议的具体目的是：1) 确定凝血酶是否可以调节已知的角膜细胞步骤 伤口愈合。培养的角膜上皮细胞、基质角质形成细胞、成纤维细胞和/或肌成纤维细胞 用凝血酶处理后，将检测凝血酶依赖性表型、细胞凋亡率、细胞总数的变化 数量、细胞分裂和迁移。2)确定凝血酶是否刺激角膜 基质细胞合成与伤口愈合有关的蛋白质。凝血酶对血管紧张素转换酶的影响 细胞因子、趋化因子、生长因子和纤溶酶原激活系统组分的合成 用实时定量RT-PCR检测凝血酶依赖的mRNA变化，并用ELISA法和/或 蛋白质水平变化的蛋白质印迹。3)确定其诱导机制。 凝血酶对角膜细胞功能和基因表达的影响它的作用机制 凝血酶诱导间质肌成纤维细胞分裂的改变和对PAI-1合成的刺激 使用凝血酶抑制剂、灭活凝血酶、凝血酶多肽、激动剂和拮抗剂测定 凝血酶敏感的蛋白水解酶激活受体和信号通路抑制物。4)确定 凝血酶和PAR-1对体内角膜创面愈合是否重要 在器官培养方面。兔和人器官培养模型和利用正常和 PAR-1缺陷小鼠将用于这些研究。

Lay Description: Wound healing in the cornea is incompletely understood; yet each year thousands of people elect corrective photorefractive surgery. Identification of mechanisms involved in wound healing will lead to the development of treatments for patients who do not heal properly. In this proposal, thrombin, a protease that generates fibrin and regulates cellular processes, will be studied. Scientific Description: The goal of this application is to test the hypothesis that thrombin is involved in corneal wound healing through cleavage of protease activated receptors and initiation of signaling pathways. Our preliminary data show the components required to convert prothrombin to thrombin and protease activated receptors mRNA in the human cornea and the ability of thrombin to alter corneal stromal cell gene expression and cell division. The thrombin inhibitor hirudin inhibits corneal epithelial wound healing. The SPECIFIC AIMS of this proposal are: 1) TO DETERMINE WHETHER THROMBIN CAN REGULATE KNOWN CELLULAR STEPS IN CORNEAL WOUND HEALING. Cultured corneal epithelial cells, stromal keratocytes,fibroblasts and/or myofibroblasts treated with thrombin will be assayed for thrombin dependent changes in phenotype, apoptosis, total cell number, cell division, and migration. 2) TO DETERMINE WHETHER THROMBIN STIMULATES CORNEAL STROMAL CELL SYNTHESIS OF PROTEINS INVOLVED IN WOUND HEALING. The effect of thrombin on cytokine, chemokine, growth factor and plasminogen activator system component synthesis will be determined using real-time RT-PCR to characterize thrombin dependent mRNA changes and ELISA and/or western blots for changes in protein levels. 3) TO DETERMINE THE MECHANISM OF INDUCTION OF THROMBIN EFFECTS ON CORNEAL CELL FUNCTION AND GENE EXPRESSION. The mechanism of thrombin induced changes in cell division of stromal myofibroblasts and stimulation of PAI-1 synthesis will be determined using thrombin inhibitors, inactivated thrombin, thrombin peptides, agonist and antagonists to thrombin sensitive protease activated receptors and signaling pathway inhibitors. 4) TO DETERMINE WHETHER THROMBIN AND PAR-1 ARE IMPORTANT FOR CORNEAL WOUND HEALING IN VIVO AND IN ORGAN CULTURE. Rabbit and human organ culture models and an in vivo model using normal and PAR-1 deficient mice will be used for these studies.