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Functional analysis of protein complex involved in neuropathic pain by proteomics

通过蛋白质组学对参与神经病理性疼痛的蛋白质复合物进行功能分析

基本信息

批准号：
15390109
负责人：
ITO Seiji
金额：
$ 8.64万
依托单位：
Kansai Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

Since human genome project is accomplished, proteomics that enables global analysis of proteins expressed in cells and tissues has been paid much attention at present. Primary afferent fibers are originated from pseudounipolar sensory neurons in dorsal root ganglia (DRG) and innervate polymodal receptors in the periphery, which are most effectively excited by noxious heat, chemical and mechanical stimuli, and transmit these impulses in the superficial dorsal horn of the spinal cord, an important site of pain processing. Nerve injury often causes intractable and chronic pain. Since neuronal plasticity has been shown to be an important component in the generation of neuropathic pain, changes in gene expression, protein expression and post-translational protein modification have been extensively studied in the DRG as well as the spinal cord. In order to elucidate the mechanisms of generation of neuropathic pain, here we attempted to identify functional molecules involved in 1) neural pola … More rity of axonal transport of proteins produced in the DRG through primary afferent fibers and 2) generation of neuropathic pain, especially NMDA receptor complex in the postsynaptic density (PSD) by use of proteomic technologies and obtained following results.1)We established large-sized two-dimensional gel electrophoresis (20 X 70 cm) and subsequent identification by MALDI-TOF-MS. We also established differential analysis of protein expression of two samples in a single gel by Ettan DIGE system.2)We found that 69 and 61 spots were at least 2-fold abundantly expressed in lumbar spinal nerve segments peripheral (P) and central (C) to the DRG respectively, among more than 800 protein spots visualized by silver staining. One of the unique spots in the P fraction was identified as an isoform of collapsin response mediator protein-2 (CRMP-2), which was decreased after nerve injury.3)We found that phosphorylation of NMDA receptor subtype NR2B increased in the PSD of spinal dorsal horn associated with neuropathic pain. At present, we have examined changes of components of NMDA receptor complex in spinal PSD of inflammatory and neuropathic pain model by proteomics. Less

自从人类基因组计划完成以来，能够对细胞和组织中表达的蛋白质进行全球分析的蛋白质组学目前受到了极大的关注。初级传入纤维起源于背根神经节(DRG)的假单极感觉神经元，支配外周的多模式感受器，在伤害性的热、化学和机械刺激下最有效地兴奋，并在脊髓浅背角传递这些脉冲，脊髓背角是疼痛处理的重要部位。神经损伤通常会导致顽固性和慢性疼痛。由于神经元的可塑性被证明是神经病理性疼痛产生的重要组成部分，基因表达、蛋白表达和翻译后蛋白修饰的变化在背根节和脊髓中得到了广泛的研究。为了阐明神经病理性疼痛的发生机制，我们试图确定参与神经Pola…的功能分子。2)利用蛋白质组学技术在突触后密度(PSD)中产生神经病理性疼痛，特别是NMDA受体复合体，并获得以下结果：1)建立了大尺寸双向凝胶电泳法(20×70 cm)，并用MALDI-TOF-MS进行鉴定。用Ettan dige系统对同一凝胶中两个样本的蛋白质表达进行了差异分析。2)银染显示的800多个蛋白质斑点中，有69个斑点在背根节周围的腰脊神经节段(P)和61个斑点(C)至少2倍丰富表达。P组分的一个独特的点被鉴定为崩溃素反应介体蛋白-2(CRMP-2)的异构体，在神经损伤后下降。3)我们发现与神经病理性疼痛相关的脊髓背角PSD中NMDA受体亚型NR2B的磷酸化增加。目前，我们已经用蛋白质组学方法研究了炎症性和神经病理性疼痛模型脊髓PSD中NMDA受体复合体成分的变化。较少