In vitro injury paradigms

体外损伤范例

• 批准号: 6396015
• 负责人: ROBERT M. SAPOLSKY
• 金额: $ 18.53万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2001-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6396015
• 关键词: Adenoviridae; Alphaherpesvirinae; BCL2 gene /protein; antioxidants; astrocytes; brain cell; cell death; cerebral ischemia /hypoxia; disease /disorder model; enzyme induction /repression; gene therapy; genetically modified animals; glia; hippocampus; laboratory mouse; neuroprotectants; occipital lobe /cortex; stress proteins; superoxide dismutase; tissue /cell culture; transfection /expression vector

The protective potential of three groups of genes will be studied in ischemia-like injury of brain injury of brain cells from cortex, hippocampus, and striatum, in primary culture. First an antioxidant strategy will be tested by over-expressing CuZn superoxide dismutase (SOD1) using herpes virus and adenoviral vectors to achieve rapid expression in neurons and astrocytes, and retroviral vectors for prolonged, stable expression in astrocytes. Whether acute expression can provide protection will be tested. If this is not protective, the effect of prolonged stable expression and the use of bicistronic vectors, to rapidly express both SOD and a downstream antioxidant vectors, to rapidly express both SOD and a downstream antioxidant enzyme, will be tested. We will test for protective effects, as well as for induction of other antioxidant enzymes. Whether protection correlates with induction of other antioxidant enzymes will be determined. Under conditions where protections seen the extent of oxygen radical production, lipid peroxidation and changes in level of glutathione will be determined. Whether this gene protects against necrotic or apoptotic forms of cell death will be determined. Whether this gene protects against necrotic or apoptotic forms of cell death will be determined, as well as the time window in which expression can still protect, since it is important to develop therapeutic strategies that are effective after insults. Second, we will study the ability of Bcl-2 expression to protect in the same injury paradigms. Oxidative status and the time window in which Bcl-2 can protect will be determined. Third we will study the ability of the inducible heat shock protein 70 (HSP70) to protect from these injury again analyzing the time window during which this gene can protect and whether it blocks apoptotic or necrotic cell death. Primary cultures are particularly useful for analyzing mechanisms of ischemic brain injury and mechanisms of protection at the cellular level. Primary cultures of neurons and glial cells and pure astrocyte cultures will be mad3e from hippocampus and striatum. Results will be compared with parallel studies carried out on primary cultures from neocortex. In addition, astrocyte cultures will be produced from transgenic mice made with different gene dosages of SOD1 or MnSOD (SOD2), including knockouts lacking these genes, to determine the importance of these enzymes for astrocyte survival of ischemia-like insults. These studies will provide fresh insight into possible mechanisms of protection by three candidate genes for anti- ischemic gene therapy tested in three brain regions.

将在原代培养的皮质、海马和纹状体脑细胞的缺血样脑损伤中研究三组基因的保护潜力。首先，将通过使用疱疹病毒和腺病毒载体过表达铜锌超氧化物歧化酶（SOD1）来测试抗氧化策略，以实现在神经元和星形胶质细胞中的快速表达，以及使用逆转录病毒载体在星形胶质细胞中实现长期、稳定的表达。急性表达是否可以提供保护将被测试。如果这不能起到保护作用，则将测试延长稳定表达和使用双顺反子载体快速表达SOD和下游抗氧化载体、快速表达SOD和下游抗氧化酶的效果。我们将测试保护作用以及其他抗氧化酶的诱导作用。将确定保护作用是否与其他抗氧化酶的诱导相关。在看到保护作用的条件下，将确定氧自由基产生的程度、脂质过氧化和谷胱甘肽水平的变化。该基因是否可以防止细胞死亡的坏死或凋亡形式将被确定。将确定该基因是否可以防止细胞死亡的坏死或凋亡形式，以及表达仍然可以提供保护的时间窗口，因为开发在损伤后有效的治疗策略非常重要。其次，我们将研究 Bcl-2 表达在相同损伤范例中的保护能力。将确定氧化状态和 Bcl-2 可以保护的时间窗口。第三，我们将研究诱导热休克蛋白 70 (HSP70) 防止这些损伤的能力，再次分析该基因可以保护的时间窗口以及它是否阻止细胞凋亡或坏死性细胞死亡。原代培养物对于分析缺血性脑损伤的机制和细胞水平的保护机制特别有用。神经元和神经胶质细胞的原代培养物以及纯星形胶质细胞培养物将从海马和纹状体中获得。结果将与对新皮质原代培养物进行的平行研究进行比较。此外，星形胶质细胞培养物将从用不同基因剂量的 SOD1 或 MnSOD (SOD2) 制成的转基因小鼠中产生，包括缺乏这些基因的敲除，以确定这些酶对于星形胶质细胞在缺血样损伤中存活的重要性。这些研究将为在三个大脑区域测试的抗缺血基因治疗的三个候选基因的可能保护机制提供新的见解。