Proteases in the Cornea

描述（由申请人提供）：Lay描述：角膜创面愈合不完全了解；然而，每年都有成千上万的人选择矫正光屈光手术。确定参与伤口愈合的机制将有助于开发治疗不能正常愈合的患者的方法。在这个提议中，凝血酶，一种产生纤维蛋白和调节细胞过程的蛋白酶，将被研究。科学描述：本应用的目的是测试凝血酶通过蛋白酶激活受体的裂解和信号通路的启动参与角膜伤口愈合的假设。我们的初步数据显示了在人类角膜中将凝血酶原转化为凝血酶和凝血酶活化受体mRNA所需的成分，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。凝血酶抑制剂水蛭素抑制角膜上皮伤口愈合。这项提议的具体目的是：1)确定凝血酶是否可以调节角膜伤口愈合的已知细胞步骤。用凝血酶处理培养的角膜上皮细胞、间质角质细胞、成纤维细胞和/或肌成纤维细胞，检测凝血酶依赖性的表型、凋亡、细胞总数、细胞分裂和迁移的变化。2)确定凝血酶是否刺激角膜基质细胞合成参与创面愈合的蛋白。凝血酶对细胞因子、趋化因子、生长因子和纤溶酶原激活物系统组分合成的影响将通过实时RT-PCR表征凝血酶依赖性mRNA的变化，以及ELISA和/或western blots检测蛋白水平的变化来确定。3)确定凝血酶诱导角膜细胞功能及基因表达的作用机制。凝血酶诱导间质肌成纤维细胞分裂变化和刺激PAI-1合成的机制将通过凝血酶抑制剂、失活凝血酶、凝血酶肽、凝血酶敏感蛋白酶激活受体的激动剂和拮抗剂以及信号通路抑制剂来确定。4)确定凝血酶和par-1在体内和器官培养角膜创面愈合中是否重要。这些研究将使用兔和人器官培养模型以及正常和PAR-1缺陷小鼠的体内模型。

DESCRIPTION (provided by applicant): 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.