Ethanol and NOS2 Gene Expression in Human Astrocytes

描述（由申请方提供）：乙醇通过以下方式改变啮齿动物iNOS活性： 改变Nos 2基因表达。目前还没有关于乙醇对 人iNOS活性。我们有来自人类A172星形细胞瘤细胞的数据， 低和高乙醇浓度对iNOS活性的双相作用。的 工作假设是乙醇对人星形胶质细胞的双相作用 iNOS活性是由于对NOS 2基因表达的双相作用。中断 NOS 2基因表达的增加， 减少iNOS的产生和随后的一氧化氮产生的变化。 然后CNS免疫应答将受到损害，导致增加的免疫应答。 易受感染和脑损伤。该项目将提供一个 长期目标的关键组成部分，旨在深入了解 酒精对中枢神经系统基因的影响与大脑疾病的病理学有关。的 具体目标是：（1）进一步确定急性和慢性 体外乙醇暴露对iNOS活性、蛋白表达和mRNA水平的影响 在正常人星形胶质细胞（NHA）和人A172星形胶质细胞瘤细胞中;和（2） 确定双相急性和慢性效应的机制 乙醇对人NOS 2基因表达的影响急性和慢性乙醇的影响 将通过评估暴露于尼古丁诱导的NOS 2表达来确定 亚硝酸盐生成、iNOS蛋白和mRNA水平。对NOS 2启动子的影响 通过用荧光素酶报告基因转染A172细胞来研究活性 基因构建体。使用删除结构将有助于识别 乙醇敏感区域，用于将来研究涉及的反式作用因子 在乙醇的作用下

DESCRIPTION (provided by applicant): Ethanol alters rodent iNOS activity by changing Nos2 gene expression. No published data exists for ethanol effects on human iNOS activity. We have data from human A172 astrocytoma cells showing a biphasic effect of low and high ethanol concentrations on iNOS activity. The working hypothesis is that the biphasic effect of ethanol on human astrocyte iNOS activity is due to a biphasic effect on NOS2 gene expression. Disruption of NOS2 gene expression by ethanol would result in either increased or decreased production of iNOS and subsequent changes in nitric oxide generation. The CNS immune response would then be compromised resulting in increased susceptibility to infection and brain damage. This project will provide a crucial component of a long-term goal designed to lend insight into the effect of alcohol on CNS genes involved in the pathology of brain disorders. The specific aims are: (1) to further characterize the effects of acute and chronic in vitro ethanol exposure on iNOS activity, protein expression, and mRNA levels in normal human astrocytes (NHA) and human A172 astrocytoma cells; and (2) to determine the mechanism underlying the biphasic acute and chronic effects of ethanol on human NOS2 gene expression. Effects of acute and chronic ethanol exposure on cytokine-induced NOS2 expression will be determined by assessing nitrite production, iNOS protein and mRNA levels. Effects on NOS2 promoter activity will be studied by transfection of A172 cells with luciferase reporter gene constructs. Use of deletion constructs will help identify ethanol-sensitive regions for future studies on trans-acting factors involved in the action of ethanol.