CHEMOKINE RECEPTOR EXPRESSION IN THE CNS

这是第二次重新应用来研究它的类型和功能 大鼠体内的趋化素受体得到了极大的改善 缩小了关注范围，提高了成功的可能性。它有三个 来自先前提议的具体目标，但规模较小 在范围内。第一个具体目标是定义 与趋化因子受体CKR2、CKR5和RBS11结合的配体。 这一目标包括两个部分。在第一部分中，他将进入HEK293 使用pcDNA3载体的细胞。他将评估该计划的成效。 Northern印迹法检测mRNA的表达。他会的 通过暴露转基因细胞或它们的细胞来确定配体结合 膜到放射性标记的MCP-1。他将决定是否 这种结合的特异性可以与各种其他CC竞争 Chomolines；将测定离解常数。他也会做 MIP-1a的CKR5也是如此。在这个目标的第二部分，他 将尝试定义跨膜信号转导的选择性 不同的细胞因子通过这些特定的受体。COS、HEK293或 K562与他将研究的大鼠趋化因子受体。这个 信令的有效性将通过确定 花生四烯酸产生，细胞内钙增加，肌醇 磷酸盐生产和cAMP积累[初步数据 表明调查员有能力做到这一点]。如果他没能发现 信号，他将使用这些细胞与人的Ga14或 在这些信元中定义了有效信令Ga15 系统。特异性受体的剂量依赖性反应 将为一些趋化因子配体建立配体。 在第二个目标中，他将检验大鼠小胶质细胞的假设 体外表达有效的Chomokine受体。在这篇文章的第一部分 目的他将研究小胶质细胞对趋化因子的反应。控制和 细胞因子(INFg或内毒素)刺激的小胶质细胞将暴露于4 CC趋化因子与由参数决定的信号转导 如上所述。细胞内信号增强的机制 将对钙进行研究。信号传递机制增强 将对细胞内钙离子进行研究。小胶质细胞预先处理 百日咳毒素(通过ADP前糖基化G-G来钝化反应) 蛋白质的阿尔法亚基)将被研究以确定 细胞内钙蓄积。如果在耗尽的钙中信令失败 中，信号源必须是外部的。这将允许调查员 要定义同时绑定多个 趋化因子对受体的作用。在目标的第二部分，水平 趋化因子受体在体外培养的小胶质细胞中的表达 将对多种细胞因子进行研究。Chomokine 对造血细胞最有效的浓度将进行比较 对小胶质细胞的作用剂量和作用时间。水平的变化 将通过整个细胞的Northern印迹来确定表达的 M RNA和/或RNA保护试验。 在最终目标中，他将使用体内系统。这将测试 小胶质细胞表达功能性Chomokine受体的假设。 这将通过特定中枢神经系统组织的原位杂交来完成 在已知可以激活小胶质细胞的明确操作之后。 有待研究的系统是面神经挤压，或称EAE。这些细胞 调控趋化因子受体将通过原位确定 当特定的细胞分类时，杂交将由 相邻晶体切片上的免疫细胞化学。就地证明意志 针对特定的趋化因子受体mRNAs和 Ga15分子的mRNA。这些在同一个版本中的表达 将定义操作后的单元格类型。

This is a second reapplication to investigate the type and function of chemoline receptors in the rat has been greatly improved by a narrowed focus and improved probability of success. It has three specific aims which come from prior proposals, but which are smaller in scope. The first specific aims are to define the specificity of the ligand binding to the chemokine receptors CKR2, CKR5 and RBS11. This aim has two parts. In the first part he will transect into HEK293 cells using the pcDNA3 vector. He will assess the effectiveness of the transfection by assaying for mRNA by Northern blotting. He will determine ligand binding by exposing transfected cells or their membranes to radiolabeled MCP-1. He will determine if the specificity of this binding can be competed with a variety of other CC chomolines; dissociation constants will be determined. He will also do the same for the CKR5 for MIP-1a. In the second part of this aim, he will attempt to define the selectivity for transmembrane signaling of various cytokines through these specific receptors. COS, HEK293, or K562 with the rat chemokine receptors he will study. The effectiveness of signaling will be assessed by determining the arachidonic acid production, intracellular calcium increased, inositol phosphate production and cAMP accumulation [preliminary data shows the ability of the investigator to do this]. If he fails to detect signaling, he will use these cells cotransfected with human ga14 or Ga15 for which effective signaling has been defined in these cell systems. The dose dependent response of the receptors for specific ligands will be established for a number of the chemokine ligands. In the second aim he will test the hypothesis that rat microglial cells express effective chomokine receptors in vitro. In the first part of this aim he will examine microglial responses to chemokines. Control and cytokine (INFg or LPS) stimulated microglial cells will be exposed to 4 CC chemokines and signal transduction determined by the parameters noted above. The mechanism of signaling increased intracellular calcium will be studies. The mechanism of signaling increases intracellular calcium will be studies. Microglia pre-treated with pertussis toxin (to blunt response by pre-ADP-gibosylating the G- protein's alpha subunit) will be studied to determine the source of intracellular calcium accumulating. If signaling fails in a Ca depleted medium, the source must be external. This will permit the investigator to define synergistic effects from the simultaneous binding of multiple chemolines to receptors. In the second part of the aims, the level of expression of chemokine receptors in cultured microglial cells exposed to a variety of cytokines will be studied. The chomokine concentrations most effective on hematopoetic cells will be compared to the dose and time of effect on microglial cells. The change in levels of expression will be determined by northern blotting of whole cell mRNA and/or by RNA protection assay. In the final aim, he will use an in vivo systems. This will test the hypothesis that microglial cells express functional chomokine receptors. This will be done by in situ hybridization on specific CNS tissues following defined manipulations known to activate microglial cells. The systems to be studies are facial nerve crush, or EAE. The cells modulating chemokine receptors will be defined by in situ hybridization while specific cells typed will be delineated by immunocytochemistry on adjacent croystat sections. In situ proves will be directed against specific chemokine receptor mRNAs and the mRNA for the Ga15 molecule. The expression of these in the same cell type following manipulation will be defined.