CARDIOCYTE RESPONSES TO HYPOXIA

描述(改编自申请人的摘要)：长期目标 这一应用是为了定义心脏细胞对 氧化还原压力，分析介导这些反应的DNA调控元件， 为外源基因在缺血心肌中的表达开辟了一条新途径。 心肌缺血对心脏施加两种主要形式的氧化还原应激 心肌细胞：1)缺氧成分，由心肌损伤的严重程度决定。 缺血；2)复氧引起的高氧应激。这两种形式的 压力会引发一系列复杂的反应。至少两个分子 对低氧的遗传反应可以区分为：快速、即时的早期 响应(几分钟到几个小时)和几个小时内发生的延迟响应 几天。原癌基因的诱导是早期反应的一部分， 糖酵解酶基因、骨骼α-肌动蛋白和内皮素的诱导 (ET-1)是晚期缺氧反应基因的例子。无论是氧气 传递氧化还原信号的感测机制和第二信使也不是 为人所知。对氧化应激的反应必然是快速的，因为 活性氧中间体具有很高的活性，可以立即氧化 细胞蛋白质和脂类不会远离它们的 一代。建议进行研究，以研究氧化还原调节 心肌细胞和血管内皮细胞中的特异性基因。这个 严重氧化还原应激对锌指转录因子的影响将是 特色化的。糖酵解酶和ET-1基因的分子分析 提出了识别低氧反应元件(HREs)和蛋白质 将他们捆绑在一起。最后，提出了体内研究的建议。 与HREs连锁的报告基因的表达。DNA将被输送到 心脏通过直接注射，心脏会受到缺血的影响，而 报告表达将与不含HRE的构建中的表达进行比较。

DESCRIPTION (adapted from the applicant's abstract): The long term goals of this application are to define molecular genetic responses of heart cells to redox stress, analyze DNA regulatory elements that mediate these responses, and define a new approach to express foreign genes in ischemic myocardium. Myocardial ischemia imposes two primary forms of redox stress on cardiac myocytes: 1) a hypoxic component determined by the severity of the ischemia, and 2) hyperoxic stress caused by reoxygenation. Both forms of stress trigger a complex series of reactions. At least two molecular genetic responses to hypoxia can be distinguished: a rapid, immediate early response (minutes to hours) and a late response that occurs over several days. Induction of proto-oncogenes is part of the early response, and induction of glycolytic enzyme genes, skeletal a-actin, and endothelin (ET-1) are examples of late hypoxia response genes. Neither the oxygen sensing mechanism nor the second messengers that relay the redox signals are known. The response to oxidative stress is obligatorily rapid since reactive oxygen intermediates are highly reactive and immediately oxidize cellular proteins and lipids without diffusing far from their sites of generation. Studies are proposed to investigate redox regulation of specific genes in cardiac myocytes and vascular endothelial cells. The impact of severe redox stress on zinc finger transcription factors will be characterized. Molecular analyses of glycolytic enzymes and ET-1 genes are proposed to identify hypoxia responsible elements (HREs) and the proteins that bind them. Finally, studies are proposed to investigate the in vivo expression of reporter genes linked to HREs. DNA will be delivered into the heart by direct injection, the heart will be subjected to ischemia, and the reporter expression will be compared with that from constructs without HREs.