MARKERS OF CNS INJURY

中枢神经系统损伤的标志

• 批准号: 2266807
• 负责人: FRANK R SHARP
• 金额: $ 18.11万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-06-01 至 1997-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2266807
• 关键词: biomarker; brain injury; calcium channel blockers; cerebral ischemia /hypoxia; gene induction /repression; gerbil /jird; glutamate receptor; hypoglycemia; immunocytochemistry; in situ hybridization; inhibitor /antagonist; laboratory rat; molecular cloning; neuroprotectants; northern blottings; nucleic acid sequence; protooncogene; stress proteins; tissue /cell culture; transfection /expression vector

The goal of this project is to understand the molecular events that occur in ischemic neurons and glia in order to develop rational strategies for preventing injury. Our previous work has shown that ischemia and other types of injury induce the c-fos immediate early gene and the hsp7O heat shock gene in rat brain and in cultured brain cells, and that induction of these genes can serve as markers of the injured cells. The current proposal will determine whether the combined use of antagonists of glutamate receptors and voltage sensitive calcium channels is required to prevent induction of c-fos mRNA, FOS protein, AP1 binding and cell injury produced in vitro by glutamate and chemical ischemia in cultured neurons and in vivo by global ischemia in gerbil hippocampal pyramidal neurons. These experiments are based upon the hypothesis that calcium entry via any route may be sufficient to induce the c-fos gene and to produce cell injury. The experiments will utilize cell culture methods and the gerbil global ischemia model. c-fos mRNA is assessed using Northern blots and in situ hybridization, Fos protein via immunocytochemistry, AP-1 binding via gel shift assays,'and cell injury via LDH in vitro and cell counts in vivo. The hsp70 heat shock gene is induced in neurons located in the penumbra of focally ischemic brain and in capillary endothelial cells but not in neurons or glia in regions of infarction. The current project will examine the induction of two other hsp70 family members, hsc7O and grp78, following focal and global ischemia. In addition, two new recently cloned stress genes, called hsr1 and hsr2, will be fully sequenced and characterized, and their expression studied following focal and global ischemia. Lastly, experiments will be performed in which retroviral vectors will be used to express the hsp70, hsc7O, and grp78 stress genes in cultured astrocytes and neurons from embryonic rat brain to determine whether expression of these genes in these cells will protect them from otherwise lethal injury. Focal ischemia is produced using the suture model in the rat and global ischemia is produced by occluding both carotids in the gerbil. Stress gene mRNA expression is examined using Northern blots and in situ hybridization. Cloning and sequencing of two new stress genes will be performed.

该项目的目的是了解发生的分子事件 在缺血性神经元和神经胶质中，以制定理性策略 防止受伤。我们以前的工作表明缺血和其他 损伤的类型诱导C-FOS立即产生早期基因和HSP7O热量 大鼠脑和培养的脑细胞中的休克基因，并诱导 这些基因可以用作受伤细胞的标志物。 当前的建议将确定是否合并 谷氨酸受体和电压敏感钙通道的拮抗剂 需要防止诱导C-FOS mRNA，FOS蛋白，AP1结合 和细胞损伤在体外由谷氨酸和化学缺血在体外产生 培养的神经元和体内由全球缺血在沙鼠海马中 金字塔神经元。这些实验是基于以下假设 通过任何途径进入钙可能足以诱导C-FOS基因，并且 产生细胞损伤。实验将利用细胞培养方法 以及Gerbil全球缺血模型。使用C-FOS mRNA使用 北印迹和原位杂交，FOS蛋白通过 免疫细胞化学，AP-1通过凝胶转移测定的结合和细胞损伤 通过体外LDH和细胞计数在体内计数。 HSP70热休克基因是在位于半阴茎的神经元中诱导的 局灶性缺血性大脑和毛细血管内皮细胞中，但不在 梗塞区域中的神经元或神经胶质。当前项目将检查 HSP70家族成员HSC7O和GRP78的诱导 遵循焦点和全球性缺血。此外，两个新的克隆 应力基因称为HSR1和HSR2，将被完全测序，并且 表征了，它们的表达在焦点和全局后进行了研究 缺血。最后，将进行逆转录病毒的实验 向量将用于表达HSP70，HSC7O和GRP78应力基因 在来自胚胎大鼠脑的培养的星形胶质细胞和神经元中 这些基因在这些细胞中的表达是否会保护它们免受 否则致命伤害。局灶性缺血是使用缝合模型产生的 在大鼠和全球缺血中，可以通过阻塞两个颈动脉来产生 沙鼠。使用Northern印迹检查应力基因mRNA表达 和原位杂交。两个新应力基因的克隆和测序 将执行。