CANNABINOID EFFECTS ON BRAIN CYTOKINES

大麻是美国使用最广泛的非法药物。 三角洲-9-四氢大麻酚(THC)，主要精神活性成分 大麻，已被证明具有免疫抑制作用，并改变 巨噬细胞和其他免疫细胞的功能活动。的目标是 这个项目是为了确定THC对细胞因子表达的影响 星形胶质细胞和小胶质细胞产生的一氧化氮。其基本原理是 检测THC对这些细胞因子和NO的影响 一些因素被认为是导致 各种神经系统疾病，包括艾滋病、痴呆症。这个 有待检验的假设是THC改变了细胞因子的表达 和/或一氧化氮通过星形胶质细胞和小胶质细胞，并且它这样做 通过大麻素受体介导的过程。星形胶质细胞和小胶质细胞 Balb/c小鼠细胞和人U373 MG星形细胞瘤细胞将作为 用体外模型评价THC对黄曲霉毒素生产的影响 白介素1、白介素6、肿瘤坏死因子和 干扰素-γ联合细菌诱导的一氧化氮(NO) 脂多糖。首先，我们会界定货柜码头处理费对 IL-1、IL-6、肿瘤坏死因子、一氧化氮的表达。免疫吸附分析将是 用于评估人和小鼠的细胞因子。一种化验方法 使用Griess试剂将被用于评估NO-2的生产。 其次，我们将确定THC改变细胞因子和/或 一氧化氮的产生。放射性标记生物合成掺入脉冲和 将进行脉冲追逐实验以确定是否表达 细胞因子和NO相关事件在转录水平受到影响， 翻译水平，或翻译后水平。第三，我们将定义 大麻素受体在改变一氧化氮和/或细胞因子中的作用 星形胶质细胞和小胶质细胞表达。的功能含义 大麻素受体在调节细胞因子表达中的作用将是 利用百日咳毒素解偶联和β(2)cAMP重组实现 实验。大麻素受体功能作用的暗示 之后将进行研究，以建立 NO和/或细胞因子产生中受体的表达和变化。 实验将包括用SR141716A选择性拮抗CB1受体， 显示立体选择性的结构活性关系 对映体对THC类似物，用特定的受体“阻断” 抗体和反义抑制大麻素信息功能。 最后，我们将进行实验，将功能链接与 受体表达。突变逆转录酶-聚合酶链式反应(MRT-PCR)， 免疫细胞化学和酶联免疫吸附试验(ELISA)将 用于区分CB1和CB2大麻素受体 表情。以这种方式，受体表达的动力学随着它们的 与巨噬细胞激活状态和NO和/或细胞因子的产生有关 将会被定义。此外，CB1和CB2受体的共同表达可以 得到承认，这将有助于解释由此产生的结果 来自功能连锁研究。

Marijuana is the most widely used illicit drug in the United States. Delta-9-tetrahydrocannabinol (THC), the major psychoactive component in marijuana, has been shown to be immunosuppressive and to alter the functional activities of macrophages and other immune cells. The goal of this project is to define the effect of THC on the expression of cytokines and nitric oxide by astrocytes and microglial cells. The rationale for examining the effect of THC on these cytokines and NO is that these factors have been implicated in playing a major role in the causation of a variety of nervous system disorders, including AIDS dementia. The hypothesis to be tested is that THC alters the expression of cytokines and/or nitric oxide by astrocytes and microglial cells and that it does so by a cannabinoid receptor-mediated process. Astrocytes and microglial cells from Balb/c mice, and human U373MG astrocytoma cells will serve as in vitro models for assessing the effect of THC on the production of interleukin-1 (IL1), interleukin-6 (IL6), tumor necrosis factor (TNF), and nitric oxide (NO) elicited by interferon-gamma plus bacterial lipopolysaccharide. First, we will define the effect of THC on the expression of IL1, IL6, TNF, and NO. Immunosorbent assays will be employed for assessment of human and murine cytokines. An assay which employs the Griess reagent will be used to assess NO-2 production. Second, we will identify the step at which THC alters cytokine and/or nitric oxide production. Radiolabel biosynthetic incorporation pulse and pulse-chase experiments will be performed to establish whether expression of cytokines and NO-related events is affected at the transcriptional, translational, or post-translational levels. Third, we will define the role of a cannabinoid receptor in altering nitric oxide and/or cytokine expression by astrocytes and microglial cells. Functional implication of a role of a cannabinoid receptor in mediating cytokine expression will be achieved using pertussis toxin uncoupling and betat(2)cAMP reconstitution experiments. Implication of a functional role for a cannabinoid receptor will be followed by studies to establish functional linkage between receptor expression and alterations in NO and/or cytokine production. Experiments will include CB1 receptor selective antagonism with SR141716A, structure activity relationships for demonstrating stereoselectivity with enantiomeric pairs of THC analogs, "blocking" of receptors with specific antibodies, and antisense inhibition of cannabinoid message functionality. Finally, we will perform experiments to relate functional linkage to receptor expression. Mutational reverse transcriptase-PCR (MRT-PCR), immunocytochemistry, and enzyme-linked immunosorbent assay (ELISA) will be performed to discriminate between CB1 and CB2 cannabinoid receptor expression. In this fashion, the kinetics of receptor expression as they relate to macrophage activation state and to NO and/or cytokine production will be defined. Furthermore, co-expression of CB1 and CB2 receptors can be recognized which would facilitate interpretation of outcomes resulting from functional linkage studies.