Functional analysis of protein complex involved in neuropathic pain by proteomics

随着人类基因组计划的完成，能够对细胞和组织中表达的蛋白质进行全局分析的蛋白质组学受到了广泛的关注。初级传入纤维起源于背根神经节（DRG）的伪单极感觉神经元，支配周围的多模态受体，这些受体最有效地受到有害的热、化学和机械刺激，并将这些冲动传递到脊髓浅表背角，这是疼痛加工的重要部位。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，基因表达、蛋白表达和翻译后蛋白修饰的变化在DRG和脊髓中得到了广泛的研究。为了阐明神经性疼痛的产生机制，本研究利用蛋白质组学技术，试图确定参与1)神经极化、DRG中产生的蛋白质通过初级传入纤维的轴突运输和2)神经性疼痛产生的功能分子，特别是突触后密度（PSD）中的NMDA受体复合物，并获得了以下结果。1)建立大尺寸双向凝胶电泳（20 X 70 cm），并进行MALDI-TOF-MS鉴定。我们还利用Ettan DIGE系统建立了两种样品在单个凝胶中的蛋白表达差异分析。2)我们发现，在800多个银染色的蛋白点中，腰脊神经节段DRG外周(P)和中央(C)分别有69个和61个蛋白点至少2倍丰富表达。P部分中一个独特的斑点被鉴定为塌陷反应介质蛋白-2 （CRMP-2）的异构体，该蛋白在神经损伤后减少。3)我们发现NMDA受体NR2B亚型磷酸化在与神经性疼痛相关的脊髓背角PSD中升高。目前，我们利用蛋白质组学方法研究了炎性和神经性疼痛模型脊髓PSD中NMDA受体复合物组分的变化。少

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