Proteases in the Cornea

角膜中的蛋白酶

基本信息

批准号：
7384416
负责人：
Sally S. Twining
金额：
$ 29.69万
依托单位：
MEDICAL COLLEGE OF WISCONSIN
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-08-01 至 2011-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7384416
关键词：
Accidents Address Adhesions Agonist Angiogenesis Inhibitors Angiostatins Antibodies Apoptosis Binding Biological Assay Caspase Cell Count Cell division Cell physiology Cells Cessation of life Characteristics Chemotaxis Cornea Corneal Injury Corneal Ulcer Data Deposition Disease Endopeptidases Enzyme-Linked Immunosorbent Assay Enzymes Epithelial Epithelial Cells Extracellular Matrix Fibrin Fibrinogen Fibroblast Growth Factor 2 Fibroblasts Fibrosis Gene Expression Goals Growth Factor Healed Hirudin Hirudins Human In Situ Nick-End Labeling Laser In Situ Keratomileusis Laser Surgery Lasers Lead Liver Lung Matrix Metalloproteinases Mediating Membrane Messenger RNA Modeling Mus Myofibroblast Operative Surgical Procedures Organ Culture Techniques Oryctolagus cuniculus PAR-1 Receptor Patients Peptide Hydrolases Peptides Phenotype Photorefractive Keratectomy Plasmin Plasminogen Plasminogen Activator Plasminogen Activator Inhibitor 1 Platelet-Derived Growth Factor Procedures Protease Inhibitor Protein Biosynthesis Proteinase-Activated Receptors Proteins Prothrombin Receptor Activation Receptor Signaling Reverse Transcriptase Polymerase Chain Reaction Role Signal Pathway Stromal Cells System Testing Thrombin Time Visual Western Blotting Wound Healing annexin A5 chemokine corneal epithelium cytokine healing in vivo in vivo Model inhibitor/antagonist keratomileusis migration research study response therapy development tissue regeneration

项目摘要

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.

外行描述：角膜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性屈光手术。识别涉及伤口愈合的机制将导致为无法正常愈合的患者开发治疗方法。在该提案中，凝血酶（一种蛋白酶）将研究产生纤维蛋白并调节细胞过程的物质。科学描述：本次活动的目标应用是为了检验凝血酶通过分裂参与角膜伤口愈合的假设蛋白酶激活受体并启动信号通路。我们的初步数据显示了组件将人类角膜中的凝血酶原转化为凝血酶和蛋白酶激活受体 mRNA 所需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。该提案的具体目标是： 1) 确定凝血酶是否可以调节角膜中已知的细胞步骤伤口愈合。培养的角膜上皮细胞、基质角膜细胞、成纤维细胞和/或肌成纤维细胞将测定用凝血酶处理的表型、细胞凋亡、总细胞的凝血酶依赖性变化数量、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参与伤口愈合的蛋白质。凝血酶的作用细胞因子、趋化因子、生长因子和纤溶酶原激活剂系统成分的合成将使用实时 RT-PCR 来表征凝血酶依赖性 mRNA 变化以及 ELISA 和/或蛋白质印迹法检测蛋白质水平的变化。 3) 确定诱导机制凝血酶对角膜细胞功能和基因表达的影响。其机制为凝血酶诱导基质肌成纤维细胞分裂的变化并刺激 PAI-1 合成使用凝血酶抑制剂、灭活凝血酶、凝血酶肽、激动剂和拮抗剂测定凝血酶敏感的蛋白酶激活受体和信号通路抑制剂。 4) 确定凝血酶和 PAR-1 在体内和体外对于角膜伤口愈合是否重要在器官培养中。兔和人体器官培养模型以及使用正常和 PAR-1 缺陷小鼠将用于这些研究。