New Gene Expression in Cell Culture Models of Ischemia

缺血细胞培养模型中的新基因表达

• 批准号: 6779018
• 负责人: David Alan Greenberg
• 金额: $ 36.06万
• 依托单位: LEGACY EMANUEL HOSPITAL AND HEALTH CENTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-15 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6779018
• 关键词: apoptosis; cerebral ischemia /hypoxia; cysteine endopeptidases; cytoprotection; functional /structural genomics; gene expression; gene targeting; genetically modified animals; glia; glucose metabolism; immunocytochemistry; laboratory mouse; mitogen activated protein kinase; mixed tissue /cell culture; neurons; neuroregulation; osteopontin; oxygen consumption; protease inhibitor; protein quantitation /detection; serine proteinases; stimulus /response; stroke; transfection /expression vector; vascular endothelial growth factors; western blottings

In Project 2, primary cortical neuronal, astroglial and mixed neuronal-astroglial cultures will be used to investigate whether proteins that show increased expression in an in vivo model of ischemic preconditioning and tolerance (Project 1) are cytoprotective in vitro. The hypothesis is that proteins showing increased expression in animal models of ischemic tolerance in vivo (Project 1) can be screened using cell culture models of ischemia and tolerance in vitro (Project 2) to identify proteins that will, in turn, protect neurons, astroglia or both from ischemia in vivo (Project 3). The specific aims are to [1] determine which candidate cytoprotective proteins show increased expression in a cerebral cortical neuronal, astroglial or mixed neuronal-astroglial cell-culture model of tolerance to oxygen and glucose deprivation (OGD); [2] investigate which candidate cytoprotective proteins protect cultured cortical neurons, astroglia or both from otherwise lethal OGD, by determining the effect on cell viability of increasing their expression or (for secreted proteins) their extracellular concentration; [3] investigate which candidate cytoprotective proteins protect cultured cortical neurons, astroglia or both from otherwise lethal OGD, by determining the effect on cell viability of reducing their expression or inhibiting their function; and [4] investigate the mechanism of action of candidate cytoprotective proteins identified in Project I and confirmed in Specific Aims 1-3 of Project 2. The most promising candidate cytoprotective proteins that emerge from Project 2 will then be tested for protective effects in vivo in Project 3. The methods used will include primary culture of rat cerebral cortical neurons and astroglia; in vitro models of cerebral ischemia, preconditioning and tolerance; cell viability assays; Western blotting; immunocytochemistry; transfection with adeno-associated virus vectors; and transgenic and gene knockout mice. The broad, long-term objective is to identify endogenous protective mechanisms in ischemia and to exploit these in the treatment of stroke.

在项目2中，将使用原代皮质神经元、星形胶质细胞和神经元-星形胶质细胞混合培养来研究在缺血预适应和耐受体内模型(项目1)中表达增加的蛋白质在体外是否具有细胞保护作用。假设在体内缺血耐受的动物模型(项目1)中表达增加的蛋白质可以使用体外缺血和耐受细胞培养模型(项目2)来筛选，以确定将反过来保护神经元和/或星形胶质细胞免受体内缺血的蛋白质(项目3)。具体目的是[1]确定哪些候选细胞保护蛋白在大脑皮层神经元、星形胶质细胞或神经元-星形胶质细胞混合培养模型中的表达增加；[2]调查 哪些候选细胞保护蛋白通过确定增加其表达或(对于分泌蛋白)细胞外浓度对细胞活力的影响来保护培养的皮质神经元、星形胶质细胞或两者免受其他致命的OGD的影响；[3]通过确定降低其表达或抑制其功能对细胞活性的影响，研究哪些候选细胞保护蛋白保护培养的皮质神经元、星形胶质细胞或两者免受否则致命的OGD的影响；和[4]研究项目I中确定并在项目2的特定目标1-3中确认的候选细胞保护蛋白的作用机制。项目2中出现的最有希望的候选细胞保护蛋白将在项目3中进行体内保护效果测试。所使用的方法将包括大鼠大脑皮质的原代培养 这些研究包括：神经元和星形胶质细胞；脑缺血、预适应和耐受的体外模型；细胞存活率分析；Western blotting；免疫细胞化学；腺相关病毒载体的导入；转基因和基因敲除小鼠。广泛的、长期的目标是确定缺血的内源性保护机制，并将其用于中风的治疗。