THROMBIN REGULATION OF CNS CELLS AFTER INJURY

中枢神经系统细胞损伤后凝血酶的调节

• 批准号: 3726118
• 负责人: DENNIS CUNNINGHAM
• 金额: --
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3726118
• 关键词: age difference; aging; amyloid proteins; apoptosis; astrocytes; cell death; cell growth regulation; cytokine; fibroblast growth factor; gene expression; glial fibrillary acidic protein; immunocytochemistry; in situ hybridization; inflammation; laboratory rat; messenger RNA; monoclonal antibody; nervous system regeneration; neural degeneration; neurons; neuroprotectants; protease inhibitor; thrombin; tissue /cell culture; vimentin

Thrombin is a multifunctional protease that regulates gene expression and process outgrowt in cultured neurons and astrocytes as well as proliferation of cultured astrocytes. Our recent studies have shown that thrombin prevents cell death in neurons cultured under hypoglycemic conditions or in the absence of growth factors. Thrombin also prevents cell death in astrocytes cultured under hypoglycemic conditions or in the presence of H202. In contrast, high concentrations of thrombin can kill both neurons and astrocytes cultured under Nnormal conditions. The effects of thrombin on these cells are receptor-mediated and can be blocked by the brain thrombin inhibitor, protease nexin-1 (PN-1). The first goal of these studies is to explore the mechanisms by which thrombin protects or kills cultured rat neurons and astrocytes. Experiments will focus on protective effects, since they occur over a broad concentration range of thrombin (10pM to 500nM) and because thrombin is one of the first molecules present at sites of trauma. Since the diverse cellular responses produced by thrombin are due to differential activation of a variety of second messenger systems, experiments will examine the role of phospholipase C, adenylyl cyclase, protein kinase G, intracellular calcium, and protein tyrosine kinase in protection and killing of neurons and astrocytes by thrombin. We will also examine the ability of thrombin to regulate certain genes, including bcl-2 and bax, which have been shown to mediate cell protection or death. Studies will also be conducted to determine if cell killing by thrombin occurs by programmed cell death or by necrosis and if it involves oxidative mechanisms. The second major goal of these studies is to test the hypothesis that thrombin might help orchestrate certain activities of astrocytes and microglia that are involved in repair/inflammatory processes, based on findings that injection of thrombin into rat brain causes an increase in reactive astrocytes and infiltration of inflammatory cells. Experiments will be conducted on cultured rat astrocy!es to determine if thrombin increases GFAP and vimentin expression as well as secretion of growth factors and cytokines such as bFGF, IL-1, IL-6 and TGF-beta. Studies will also be carried out to determine if thrombin is chemotactic for microglia and if it stimulates them to proliferate, to phagocytose particles or to secrete cytokines. The final goal of this project is to conduct in vivo experiments to evalute results obtained from studies on cultured cells. In collaboration with Dr. Cotman and his colleagues, we will administer the thrombin inhibitor PN-1 to excitotoxic lesions in rat brain that range from threshold to severe. This will enable us to examine effects of thrombin inhibition on cell death and repair processes. In collaboration with Dr. Gall, prothrombin, thrombin receptor and PN-1 mRNA will be examined by in situ hybridization in specific regions of rat brain as a function of aging. These studies will test the hypothesis that age-related chanaes in these molecules miaht compromise the abilitv to respond to certain kinds of stress or trauma.

凝血酶是一种多功能蛋白酶，其调节基因表达， 在培养的神经元和星形胶质细胞中， 培养的星形胶质细胞的增殖。 我们最近的研究表明， 凝血酶阻止低血糖下培养的神经元细胞死亡 条件下或在没有生长因子的情况下。 凝血酶还可以防止 在低血糖条件下培养的星形胶质细胞中或 H2 O2的存在。 相反，高浓度的凝血酶可以杀死 神经元和星形胶质细胞均在正常条件下培养。 的 凝血酶对这些细胞的作用是受体介导的， 被脑凝血酶抑制剂蛋白酶连接蛋白-1（PN-1）阻断。 这些研究的第一个目标是探索 凝血酶保护或杀死培养的大鼠神经元和星形胶质细胞。 实验将侧重于保护作用，因为它们发生在一个 凝血酶的浓度范围宽（10 pM至500 nM）， 是最早出现在创伤部位的分子之一以来 凝血酶产生的不同细胞反应是由于不同的 激活各种第二信使系统，实验将 研究磷脂酶C，腺苷酸环化酶，蛋白激酶G， 细胞内钙和蛋白酪氨酸激酶的保护作用， 凝血酶杀死神经元和星形胶质细胞。我们亦会研究 凝血酶调节某些基因的能力，包括bcl-2和bax， 它们被证明可以介导细胞保护或死亡。 研究将 也可以进行测定凝血酶是否杀死细胞， 程序性细胞死亡或坏死，如果它涉及氧化 机制等 这些研究的第二个主要目标是检验假设， 凝血酶可能有助于协调星形胶质细胞的某些活动， 参与修复/炎症过程的小胶质细胞，基于 研究发现，将凝血酶注射到大鼠脑中会导致 反应性星形胶质细胞和炎性细胞浸润。 实验 将在培养的大鼠星形胶质细胞上进行！以确定凝血酶是否 增加GFAP和波形蛋白表达以及生长分泌 因子和细胞因子如bFGF、IL-1、IL-6和TGF-β。 研究将 也可以确定凝血酶是否对小胶质细胞具有趋化性 如果它刺激它们增殖，吞噬颗粒， 分泌细胞因子。 该项目的最终目标是进行体内实验， 评价培养细胞研究的结果。 合作 与科特曼医生和他的同事们一起，我们将注射凝血酶 PN-1抑制剂对大鼠脑中兴奋性毒性损伤的影响范围从 阈值为严重。 这将使我们能够检查凝血酶的影响， 抑制细胞死亡和修复过程。 与博士合作。 Gall、凝血酶原、凝血酶受体和PN-1 mRNA将通过在 大鼠脑特定区域的原位杂交作为功能 衰老这些研究将检验以下假设： 这些分子可能会损害对某些类型的 压力或创伤。