Microarray analysis of neuronal ischemic preconditioning

描述(由申请人提供)： 脑缺血预适应(PC)是指各种刺激本身不会引起神经元损伤，但却能显著降低大脑对随后发生的致命性缺血的易感性。PC刺激产生的缺血耐受似乎需要新的基因转录和蛋白质合成。已经确定了启动PC反应的多种细胞内途径，但最终负责的基因仍然难以捉摸。微阵列分析可以快速识别和半定量大量的基因，这使其成为研究PC等复杂机制的理想选择。这项拟议工作的假设是，微阵列分析将允许识别PC刺激上调或下调的基因，并最终授予缺血耐受性。由于许多基因是在有害刺激后进行调节的，其中大多数与PC无关，因此挑战是将相关基因与那些仅仅是附属现象的基因区分开来。在应用PC刺激的过程中，药物阻断PC级联可以帮助过滤相关的外在现象基因。这项工作的具体目标如下：1)鉴定其转录受亚致死性缺血调控的基因，并通过使用PC的药物抑制物分离与PC相关的基因和与PC相关的基因；2)在预适应和非预适应动物中鉴定其在致死性脑缺血损伤后转录调控的基因；3)建立可能参与介导PC的候选基因库；4)确定PC候选基因，并通过RT-PCR、免疫组织化学和Western blotting确认它们的调控；5)通过在体内对已鉴定基因进行药理学调控，以确定已鉴定基因与PC的相关性，进而确定这种调控对神经元结局的影响。这项拟议研究的最终目标是确定神经元内源性的神经保护程序，并在有脑缺血风险的患者中唤起这些程序。这项研究的一个重大好处将是确定新的治疗靶点，未来的药物开发可以在此基础上进行。

DESCRIPTION (provided by applicant): Ischemic preconditioning (PC) is a phenomenon whereby various stimuli, which themselves do not cause neuronal injury, will significantly reduce the vulnerability of the brain to subsequent lethal ischemia. Ischemic tolerance conferred by a PC stimulus appears to require new gene transcription and protein synthesis. Multiple intracellular pathways have been identified that initiate the PC response, but the genes ultimately responsible remain elusive. Microarray analysis permits the rapid identification and semi-quantification of a large number of genes, making it ideal for studying complex mechanisms such as PC. The hypothesis of the proposed work is that microarray analysis will permit the identification of genes upregulated or downregulated by the PC stimuli and ultimately confer ischemic tolerance. Because many genes are modulated following a noxious stimulus, most of which are not involved in PC, the challenge is to separate relevant genes from those that are mere epiphenomena. Pharmacologic blockade of the PC cascade during application of the PC stimulus can help filter relevant from epiphenomena genes. The following are the specific aims of the proposed work: 1) To identify the genes whose transcription is modulated by sublethal ischemia and separate genes relevant to PC from those that are epiphenomena by the use of pharmacologic inhibitors of PC; 2) To identify the genes whose transcription is modulated following a lethal ischemic insult in preconditioned versus non-preconditioned animals; 3) To create a pool of gene candidates with potential involvement in mediating PC; 4) To determine the PC candidate genes and to confirm their modulation by rt-PCR, immunohistochemistry, and Western blotting; 5) To confirm the relevance of the identified genes to PC by pharmacologically modulating the genes in vivo and then to determine the effect of this modulation on neuronal outcome. The ultimate goal of the proposed research is to identify neuroprotective programs endogenous to neurons and to evoke these programs in patients who are at risk for cerebral ischemia. A substantial benefit of the research will be the identification of novel therapeutic targets upon which future drug development can be based.