大鼠背根神经节持续受压后差异蛋白质表达分析

关键词：蛋白质组学  神经病理性疼痛  持续压迫背根神经节  生物质谱

Keywords:proteomics  neuropathic pain  chronic compression of dorsal root ganglion  mass spectrometry

摘要：

      摘要 目的：使用蛋白质组学方法筛查大鼠背根神经节（DRG）持续受压（CCD）后差异表达的蛋白质，特别是筛查与疼痛和组织损伤相关的蛋白。 方法：78只Wister大鼠随机分为正常组和CCD组，建立CCD模型后，测量行为学指标。28d后处死大鼠，从DRGs中提取蛋白，进行双向电泳分离蛋白，找出差异表达蛋白，使用基质辅助激光解析电离飞行时间质谱分析得到其肽指纹图谱（PMF），进行鉴定。使用Western blotting和实时RT-PCR验证部分差异蛋白。 结果：CCD明显降低机械痛阈和热辐射刺激缩爪反应潜伏期。正常组和CCD组98个蛋白点的表达存在明显差异，成功鉴定出15种蛋白，其中8种蛋白的表达在CCD组下调，7种蛋白表达上调(其中1种蛋白只存在于CCD组)。验证显示与质谱分析的结果相符。 结论：以差异蛋白质组学的方法分析CCD对DRG蛋白质表达的影响，鉴定出15种差异表达的蛋白。其中膜联蛋白A2、p11和蛋白激酶Cε（PKCε）蛋白表达的上调，可能参与了神经性疼痛的发生过程。三磷酸甘油醛脱氧酶（GAPDH）的上调或许参与神经元凋亡，热休克蛋白70（HSP70）的表达上调或许与神经保护有关。

Abstract:

      Abstract Objective:To identify the differential protein expressions related to neuropathic pain and neuroprotection in the dorsal root ganglion (DRG)after chronic compression of DRG (CCD) in rats. Method: Seventy-eight Wister rats were randomly divided into normal group and CCD group. After CCD model was established, behavior measurements were administered in rats. Proteins and peptides were separated by two-dimensional gel electrophoresis(2DE). The differentially expressed proteins were fownd, and the peptide mass fingerprint(PMF) of protein spots were and identified by matrix assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) to study differential proteomic expressions in the L4 and L5 DRGs. Western blotting and real time RT-PCR were used to verity a part of differential proteins. Result: Mechanical withdrawal threshold and thermal drawal latency reduced significantly in CCD rats. Over 400 protein spots were visualized in normal group and CCD group respectively. A total of 98 spots were differentially expressed in these two groups and 15 spots were identified by MS analysis, of which, 8 down-regulated in CCD group and 7 up-regulated(only 1 detected in CCD group). The results of Western blotting and real-time quantitative RT-PCR experiments showed consistent with the data of proteomic analysis. Conclusion: With proteomic analysis of differential proteins, the influence of CCD on the proteins expressions in DRG and the molecular process in DRG underlying neuropathic pain were determined. CCD was associated with the up-regulation of annexin A2 and protein kirase Cε(PKCε) and their related genes. The up-regulation of glyceraldehyde-3-phosphate-dehydrogenase(GAPDH) and heat shock protein 70(HSP70) suggested that concurrent processes of nervous injury and neuroprotection in the course of neuropathic pain were existed.