Ethanol and NOS2 Gene Expression in Human Astrocytes

描述(申请人提供)：乙醇通过以下方式改变啮齿类动物的iNOS活性 改变NOS2基因的表达。目前还没有已发表的关于乙醇对 人类诱导型一氧化氮合酶活性。我们有来自人类A172星形细胞瘤细胞的数据显示 低、高乙醇浓度对iNOS活性的双相影响。这个 工作假说是乙醇对人星形胶质细胞的双相效应 INOS活性是由于NOS2基因表达的双相作用所致。颠覆 乙醇对nos2基因表达的抑制作用 诱导型一氧化氮合酶的生成减少，随后一氧化氮的生成发生变化。 中枢神经系统的免疫反应会受到损害，从而导致 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，旨在深入了解影响 酒精对参与大脑疾病病理的中枢神经系统基因的影响。这个 具体目标是：(1)进一步说明急性和慢性疾病的影响 体外酒精暴露对诱导型一氧化氮合酶活性、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星形细胞瘤细胞中； 确定急性和慢性两相效应的潜在机制 乙醇对人NOS2基因表达的影响。急性和慢性酒精的影响 暴露于细胞因子诱导的NOS2表达将通过评估 亚硝酸盐生成、iNOS蛋白和mRNA水平。对NOS2启动子的影响 通过将荧光素酶报告基因导入A172细胞来研究活性 基因结构。删除结构的使用将有助于识别 乙醇敏感区域未来涉及的反式作用因子研究 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.