PROTEASES AND CORNEAL ULCERATION

蛋白酶和角膜溃疡

• 批准号: 2160758
• 负责人: Sally S. Twining
• 金额: $ 17.26万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-07 至 1997-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2160758
• 关键词: alpha 1 antitrypsin; biological signal transduction; cornea ulcer; cycloheximide; endopeptidases; enzyme biosynthesis; enzyme induction /repression; eye injury; fibroblast growth factor; human tissue; immunoprecipitation; inflammation; interferon gamma; interleukin 1; interleukin 2; interleukin 6; messenger RNA; northern blottings; nuclear runoff assay; organ culture; polymerase chain reaction; protease inhibitor; retinoids; transforming growth factors; zymogens

Corneal ulceration occurs when the balance between proteinase and inhibitors in the cornea is disrupted. Understanding the means by which the cornea controls proteinase inhibitor levels is critical for the development of ways to prevent corneal ulceration, promote corneal healing and to maintain and/or restore transparency. We have shown that the cornea contain and synthesizes alpha1-proteinase inhibitor (alpha1- antitrypsin), alpha1-antichymotrypsin and alpha2-macroglobulin. Further, alpha1-proteinase synthesis is stimulated by the inflammatory cytokine interleukin-1Beta (IL-1Beta), basic fibroblast growth factor and retinol. The long term goal is to characterize, at the biochemical level, the process of corneal ulceration by identifying not only the proteinase involved but other factors influencing the degradation process. The general and specific hypotheses are the following: The cornea has the ability to respond to an injury and/or inflammation by the synthesis of molecules which protect the cornea thereby minimizing damage and promoting healing. Given that the cytokine IL-1Beta regulates the response of cells to injury and inflammation, we hypothesize that proteinase inhibitor synthesis in the cornea is induced by IL-1Beta as part of a coordinated early response to injury/and or inflammation. This hypothesis will be tested using both a human corneal organ culture system and a human stromal cell culture system. The specific aims of this proposal are the following: 1. To determine if IL-1Beta selectively induces the synthesis of alpha1-proteinase inhibitor by the cornea or whether it coordinates an increase in other proteinase inhibitors, other protective proteins or target proteinase in the same time frame. Synthesis of the proteins will be assays will be used to assess the relative levels of proteinase synthesized by the cornea. 2. To determine the effects of IL-1Beta function modulators on the synthesis of alpha1-proteinase inhibitor and other corneal proteinase retinoid, IL- 6, TGF-Beta, INFgamma, IL-2 and bFGF on the effects of IL-1Beta on proteinase inhibitor synthesis. 3. To determine the mechanism by which IL-1Beta and its modulators regulate the levels of alpha1-proteinase inhibitor and other proteinase inhibitors in the cornea. Inhibitors and specific agonists will be used to elucidate which signal transduction pathways are important. The effect of IL-1Beta and its modulators on the steady state mRNA levels of the inhibitors will be determined by Northern analysis. The rates of mRNA synthesis will be determined using a generated will be determined using PCR methodology. Finally the synthesis and stability of the protein product will be assayed in pulse and pulse chase experiments.

当蛋白酶和蛋白酶之间的平衡 角膜中的抑制剂被破坏。 了解哪种手段 角膜控制蛋白酶抑制剂水平对于 开发防止角膜溃疡，促进角膜的方法 治愈，维持和/或恢复透明度。 我们已经表明 角膜包含并合成α1-蛋白酶抑制剂（alpha1-- 抗胰蛋白酶），α1-抗抗胰蛋白酶和α2-巨球蛋白。 更远， 炎症细胞因子刺激α1-蛋白酶合成 白介素1Beta（IL-1Beta），基本成纤维细胞生长因子和视黄醇。 长期目标是在生化层面表征 角膜溃疡的过程不仅鉴定蛋白酶 涉及但其他影响降解过程的因素。 这 一般和特定的假设如下：角膜具有 能够通过合成来应对伤害和/或炎症 保护角膜的分子，从而最大程度地减少损害和 促进康复。 鉴于细胞因子IL-1Beta调节 细胞对损伤和炎症的反应，我们假设 角膜中蛋白酶抑制剂的合成由IL-1Beta诱导为 对损伤/或炎症的早期反应的一部分。 这 假设将使用人角膜器官培养系统进行检验 和人类基质细胞培养系统。 该提案的具体目的是：1。确定是否是否 IL-1Beta选择性诱导α1-蛋白酶抑制剂的合成 通过角膜或是否协调其他蛋白酶的增加 抑制剂，其他保护蛋白或同一靶蛋白酶 大体时间。蛋白质的合成将是分析的 评估由角膜合成的蛋白酶的相对水平。 2。 确定IL-1BETA函数调节器对合成的影响 α1-蛋白酶抑制剂和其他角膜蛋白酶类维生素类似的IL- 6，TGF-beta，Infgamma，IL-2和BFGF对IL-1Beta对影响的影响 蛋白酶抑制剂合成。 3。确定该机制 IL-1BETA及其调节剂调节α1-蛋白酶的水平 角膜中的抑制剂和其他蛋白酶抑制剂。 抑制剂和 特定的激动剂将用于阐明哪些信号转导 途径很重要。 IL-1Beta及其调节剂对 抑制剂的稳态mRNA水平将由北部确定 分析。 mRNA合成的速率将使用 生成的将使用PCR方法确定。 最后 蛋白质产物的合成和稳定性将在脉冲中分析 和脉搏追逐实验。