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 研究基因产品的神经杀灭或神经保护作用 它的表达因脑缺血而改变，如项目中所确定的 1.长期目标是确定内生机制 可在治疗中利用的神经元死亡和神经保护 中风的可能性。这些假设是：(A)一些相同的基因产物，其 脑缺血改变在体内的表达也受到类似的影响 在细胞培养模型中；(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) 基因表达改变对细胞内神经元损伤的修复能力 培养可以预测体内表达改变的效果。具体的 目的是：(1)确定哪一种候选具有神经毒性和神经保护作用 基因产物在神经细胞培养模型中的表达发生变化 缺血和细胞死亡；(2)研究哪些基因产物表现出 表达增加具有神经毒性或神经保护功能 作用，使用反义寡核苷酸(ODN)来阻断其 表达；以及(3)研究哪些基因产物表现出减少 表达具有功能效应，利用病毒载体增强其功能 表情。将使用北部和西部分析来检测 候选基因和基因产物的表达，以及阿拉马蓝 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--葡萄糖低氧 皮层神经元培养中的剥夺、兴奋性氨基酸暴露 皮层神经元培养及K/-戒断诱导的细胞凋亡 将采用小脑颗粒细胞培养。硫代磷 反义寡核苷酸将被用于抑制单链和单链的翻译 双突变复制缺陷型HSV载体将用于 过度表达，候选基因产品。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，在项目中 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.