NEW GENE EXPRESSION IN CELL CULTURE MODELS

项目2：缺血细胞培养模型中的新基因表达 在项目2中，原代神经元细胞培养模型将用于 研究基因产物的神经杀伤或神经保护作用 其表达因脑缺血而改变，如在Project 1.长期目标是确定 神经元死亡和神经保护，可用于治疗 中风这些假设是：（A）一些相同的基因产物， 在体内，脑缺血改变了表达， （B）这些模型可用于确定 基因产物影响缺血神经元的存活或死亡;（C） 改变基因表达以修饰细胞中神经元损伤的能力 培养可以预测体内表达改变的影响。具体 目的是：（1）确定哪种候选的神经杀灭和神经保护 基因产物在神经元细胞培养模型中显示改变的表达 缺血和细胞死亡;（2）研究哪些基因产物表现出 增加表达具有神经杀伤或神经保护功能 作用，使用反义寡脱氧核苷酸（ODNs）来阻断其 （3）研究哪些基因产物表现出降低的表达水平。 表达具有功能效应，使用病毒载体来增强其 表情将使用北方和西方分析来检测 候选基因和基因产物的表达，以及阿利兰 荧光和乳酸脱氢酶将用于定量 神经毒性一系列细胞培养模型-含糖缺氧 皮层神经元培养物中的剥夺， 皮质神经元培养，以及K+/-戒断诱导的细胞凋亡。 小脑颗粒细胞培养。代磷酸酯 反义ODNs将用于抑制翻译，和单-和 双突变复制缺陷型HSV载体将用于 过量表达候选基因产物。其中最有希望的将是 在体内测试，使用反义和基因转移技术， 3.

Project 2: New Gene Expression in Cell Culture Models of Ischemia In Project 2, primary neurons cell culture models will be used to investigate the neurocidal or neuroprotective effects of gene products whose expression is altered by cerebral ischemia, as identified in Project 1. The long-term objective is to identify endogenous mechanisms of neuronal death and neuroprotection that can be exploited in the treatment of stroke. These hypothesis are: (A) some of the same gene products whose expression is altered by cerebral ischemia in vivo are affected similarly in cell culture models; (B) these models can be used to determine which gene products influence whether ischemic neurons survive or die; (C) the ability of altered gene expression to modify neuronal injury in cell culture can predict effects of altered expression in vivo. The specific aims are to: (1) determine which candidate neurocidal and neuroprotective gene products show altered expression in cell culture models of neuronal ischemia and cell death; (2) investigate which gene products exhibiting increased expression have neurocidal or neuroprotective functional effects, using antisense oligodeoxynucleotides (ODNs) to block their expression; and (3) investigate which gene products exhibiting decreased expression have functional effects, using viral vectors to enhance their expression. Northern and Western analysis will be used to detect expression of candidate genes and gene products, and Alamar blue fluorescence and lactate dehydrogenase will be used to quantify neurotoxicity. A spectrum of cell culture models-hypoxia with glucose deprivation in cortical neuron cultures, excitatory amino acid exposure in cortical neuron cultures, and K+/- withdrawal-induced apoptosis in cerebellar granule cell cultures-will be employed. Phosphorothioate antisense ODNs will be used to inhibit translation of, and single- and double-mutant replication-defective HSV vectors will be used to overexpress, candidate gene products. The most promising of these will be tested in vivo, using antisense and gene transfer techniques, in Project 3.