Microarray analysis of neuronal ischemic preconditioning

描述（由申请人提供）： 缺血预处理（Ischemic preconditioning，PC）是一种本身不引起神经元损伤的各种刺激能显著降低脑对随后致死性缺血的脆弱性的现象。PC刺激所赋予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。微阵列分析允许快速识别和半定量的大量基因，使其成为研究复杂的机制，如PC的理想选择。这项工作的假设是，微阵列分析将允许识别由PC刺激上调或下调的基因，并最终赋予缺血耐受性。由于许多基因在有害刺激后受到调节，其中大多数与PC无关，因此挑战在于将相关基因与那些仅仅是附带现象的基因分开。在PC刺激的应用过程中PC级联的药理学阻断可以帮助从表观现象基因中筛选相关基因。本研究的具体目的是：1）鉴定亚致死性缺血后转录受调节的基因，并通过使用PC的药理学抑制剂将PC相关基因与那些作为副现象的基因分离; 2）鉴定预处理和非预处理动物中致死性缺血损伤后转录受调节的基因; 4）通过RT-PCR、免疫组化和Western blotting确定PC候选基因并证实其调节作用;第五章）为了通过在体内对基因进行间接调节来确认所鉴定的基因与PC的相关性，然后确定这种调节对PC的影响，神经元结果。这项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。这项研究的一个实质性好处将是确定新的治疗靶点，未来的药物开发可以基于此。

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.