In vitro injury paradigms

在原代培养中，将研究三组基因在脑缺血样损伤中对皮层、海马和纹状体脑细胞损伤的保护作用。首先，将通过使用疱疹病毒和腺病毒载体过表达CuZn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，并通过逆转录病毒载体在星形胶质细胞中实现长期稳定表达。尖锐的表达是否能起到保护作用，还有待检验。如果这不是保护性的，延长稳定表达和使用双顺反子载体的效果将被测试，以快速表达SOD和下游抗氧化剂载体，快速表达SOD和下游抗氧化物酶。我们将测试保护作用，以及其他抗氧化酶的诱导。保护是否与其他抗氧化酶的诱导相关将被确定。在保护的条件下，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。这种基因是否能防止细胞死亡的坏死性或凋亡性，将会被确定。这种基因是否可以防止细胞死亡的坏死性或凋亡性，以及表达仍然可以保护的时间窗口将被确定，因为开发在侮辱后有效的治疗策略是重要的。其次，我们将研究在相同的损伤模式下，Bcl-2表达的保护能力。氧化状态和Bcl2保护作用的时间窗将被确定。第三，我们将再次研究可诱导热休克蛋白70(HSP70)对这些损伤的保护能力，分析该基因可以保护的时间窗口，以及它是否阻止细胞的凋亡或坏死。原代培养对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和纯星形胶质细胞的培养将来自海马区和纹状体。结果将与对新皮质原代培养进行的平行研究进行比较。此外，还将从含有不同SOD1或MnSOD(SOD2)基因剂量(包括缺失这些基因的敲除)的转基因小鼠中培养星形胶质细胞，以确定这些酶对星形胶质细胞在缺血样侮辱中存活的重要性。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可能的保护机制提供新的见解。

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.