CANNABINOID EFFECTS ON BRAIN CYTOKINES

大麻是美国使用最广泛的非法药物。 Delta-9-tetrahydrocannabinol（THC）是大麻中的主要精神活性成分， 大麻，已被证明是免疫抑制和改变 巨噬细胞和其他免疫细胞的功能活动。 的目标 本课题旨在明确THC对细胞因子表达的影响， 和一氧化氮。 的理由 检查THC对这些细胞因子和NO的影响， 因素已被牵连在发挥主要作用的因果关系 各种神经系统疾病，包括艾滋病痴呆症。 的 有待检验的假设是THC改变了细胞因子的表达 和/或一氧化氮的星形胶质细胞和小胶质细胞， 通过大麻素受体介导的过程。 星形胶质细胞和小胶质 来自Balb/c小鼠的细胞和人U373 MG星形细胞瘤细胞将用作 用于评估THC对产生THC的影响的体外模型 白细胞介素-1（IL 1）、白细胞介素-6（IL 6）、肿瘤坏死因子（TNF）和 一氧化氮（NO）诱导的干扰素-γ加细菌 脂多糖 首先，我们会界定货柜码头处理费对 IL 1、IL 6、TNF和NO的表达。 用于评估人和鼠细胞因子。 的测定物 使用Griess试剂将用于评估NO-2的产生。 第二，我们将确定THC改变细胞因子和/或 一氧化氮生成 放射性标记生物合成掺入脉冲和 将进行脉冲追踪实验以确定是否表达 细胞因子和NO相关事件的影响是在转录， 翻译或翻译后水平。 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的表达。 的功能含义 大麻素受体在介导细胞因子表达中的作用将是 使用百日咳毒素解偶联和betat（2）cAMP重建实现 实验 大麻素受体功能作用的意义 随后将进行研究，以建立 受体表达和NO和/或细胞因子产生的改变。 实验将包括CB 1受体与SR 141716 A的选择性拮抗作用， 用于证明立体选择性的构效关系 THC类似物的对映体对，“阻断”具有特异性的受体， 抗体和大麻素信息功能的反义抑制。 最后，我们将进行实验，将功能连接与 受体表达 突变逆转录酶-PCR（MRT-PCR）， 免疫细胞化学和酶联免疫吸附试验（ELISA）将 用于区分CB 1和CB 2大麻素受体 表情 以这种方式，受体表达的动力学，因为它们 涉及巨噬细胞活化状态和NO和/或细胞因子产生 将被定义。 此外，CB 1和CB 2受体的共表达可 这将有助于解释结果， 从功能联系研究中。

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.