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，主要神经元细胞培养模型将用于 研究基因产物的神经保护作用或神经保护作用 在项目中确定的脑缺血改变了其表达 1。长期目标是确定内源性机制 可以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 体内的脑缺血改变了表达。 在细胞培养模型中； （b）这些模型可用于确定哪个 基因产物会影响缺血性神经元生存还是死亡。 （c） 基因表达改变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）确定哪种候选神经胶质性和神经保护作用 基因产物在神经元细胞培养模型中表现出改变 缺血和细胞死亡； （2）研究哪些基因产物表现出 表达增加具有神经保护功能或神经保护功能 效果，使用反义寡脱氧核苷酸（ODN）阻止其 表达; （3）调查哪些基因产物降低 表达具有功能效应，使用病毒向量来增强其 表达。北方和西方分析将用于检测 候选基因和基因产物的表达和Alamar Blue的表达 荧光和乳酸脱氢酶将用于量化 神经毒性。葡萄糖的细胞培养模型 - 催眠素 皮质神经元培养物的剥夺，兴奋性氨基酸暴露 皮质神经元培养物和K +/-戒断诱导的凋亡 小脑颗粒细胞培养物将使用。磷酸盐剂 反义ODN将用于抑制单一和单一和单一的翻译 双突复制缺陷性HSV向量将用于 过表达，候选基因产品。这些最有希望的是 在项目中使用反义和基因转移技术在体内测试 3。