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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

自人类基因组计划完成以来，蛋白质组学作为一种能够对细胞和组织中表达的蛋白质进行全面分析的技术受到了广泛的关注。初级传入纤维起源于背根神经节（dorsal root ganglia，DRG）中的假单极感觉神经元，支配外周的多模态感受器，这些感受器最有效地被有害的热、化学和机械刺激兴奋，并将这些冲动传递到脊髓背角浅层，这是疼痛处理的重要部位。神经损伤常引起顽固性和慢性疼痛。由于神经元可塑性已被证明是神经性疼痛产生的重要组成部分，因此背根节和脊髓中基因表达、蛋白质表达和翻译后蛋白质修饰的变化已被广泛研究。为了阐明神经病理性疼痛的发生机制，我们试图鉴定1）神经极性蛋白（neuropathic pain，NPA）， ...更多信息 DRG中产生的蛋白质通过初级传入纤维的轴突运输的多样性和2）神经性疼痛的产生，尤其是NMDA受体复合物在突触后致密区（PSD）的表达，获得了如下结果：1）建立了大片段双向凝胶电泳技术（20 X 70 cm），随后通过MALDI-TOF-MS进行鉴定。我们还通过Ettan DIGE系统建立了两种样品在单一凝胶中蛋白表达的差异分析。2）我们发现，在银染显示的800多个蛋白质点中，分别有69个和61个点在DRG的腰脊神经节段外周（P）和中央（C）中至少2倍的丰富表达。P组分中的一个独特的点被鉴定为神经肽反应介导蛋白-2（CRMP-2）的亚型，其在神经损伤后减少。3）我们发现，在与神经病理性疼痛相关的脊髓背角PSD中，NMDA受体亚型NR 2B的磷酸化增加。目前，我们已经用蛋白质组学的方法研究了NMDA受体复合物组分在炎症性和神经病理性疼痛模型脊髓PSD中的变化。少