NEW GENE EXPRESSION IN CELL CULTURE MODELS

细胞培养模型中的新基因表达

• 批准号: 6494879
• 负责人: David Alan Greenberg
• 金额: $ 38.25万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至 2002-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6494879
• 关键词: antisense nucleic acid; apoptosis; cerebral ischemia /hypoxia; disease /disorder model; excitatory aminoacid; gene induction /repression; neural degeneration; neurogenetics; neurons; neuropharmacology; oligonucleotides; tissue /cell culture

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.

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灭或神经保护作用 正如项目中所确定的，其表达会因脑缺血而改变 1. 长期目标是找出内生机制 可在治疗中利用的神经元死亡和神经保护 中风。这些假设是： (A) 一些相同的基因产物，其 体内脑缺血会改变表达，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哪些 基因产物影响缺血神经元的存活或死亡； （三） 改变基因表达改变细胞神经元损伤的能力 培养可以预测体内表达改变的影响。具体的 目的是：（1）确定候选的神经杀灭剂和神经保护剂 基因产物在神经元细胞培养模型中表现出表达改变 缺血和细胞死亡； (2)调查哪些基因产物表现出 表达增加具有神经杀伤或神经保护功能 使用反义寡脱氧核苷酸 (ODN) 来阻断其效应 表达; (3) 研究哪些基因产物表现出下降 表达具有功能效应，使用病毒载体来增强其 表达。北部和西部分析将用于检测 候选基因和基因产物的表达，以及阿拉玛蓝 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-葡萄糖缺氧 皮质神经元培养中的剥夺，兴奋性氨基酸暴露 皮质神经元培养物和 K+/- 戒断诱导的细胞凋亡 将采用小脑颗粒细胞培养物。硫代磷酸酯 反义 ODN 将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 过表达，候选基因产物。其中最有希望的是 在 Project 中使用反义和基因转移技术进行体内测试 3.