趋化因子MCP-1对辣椒素受体（TRPV1）功能的易化作用及其机制研究

发布时间：2018-05-03 01:05

本文选题：神经病理性疼痛 + 趋化因子MCP-1　；参考：《第二军医大学》2011年硕士论文

【摘要】：单核细胞趋化蛋白-1 (monocyte chemoattractant protein-1;MCP-1)属于CC类趋化因子。近年来的研究表明,在神经病理性疼痛、炎症或其他病理性变等情况下,趋化因子MCP-1及其受体CCR2在神经元和胶质细胞上表达上调,并能直接引起痛觉神经元兴奋性持续增高、伤害性感受器基因表达改变。MCP-1被认为是疼痛家族的一个新成员,是一个重要的导致疼痛发生的因子。辣椒素受体TRPV1在背根神经节、三叉神经节等中小型神经元的C类无髓纤维或Aδ神经纤维等疼痛感受器上大量表达,是配体门控的非选择性阳离子通道。越来越多的研究表明,TRPV1在痛觉的产生及痛觉敏感性增强的病理过程中扮演着重要角色。为了进一步研究MCP-1对TRPV1疼痛效应的调控机制,本课题在大鼠慢性压迫背根神经节(CCD)的模型上,应用Real-time PCR和免疫组织化学染色明确CCD背根神经节细胞上,MCP-1受体CCR2与TRPV1等的表达与分布;用Western blot的方法对CCD、CCI以及SNL三种神经损伤模型上CCR2在DRG中的表达进行了定量分析;应用行为学测试方法观察MCP-1是否易化CCD大鼠上TRPV1介导的痛觉行为,应用全细胞膜片钳及钙离子成像技术,观察了MCP-1对CCD大鼠DRG神经元上TRPV1通道的调控作用及可能的细胞内信号途径。主要研究结果如下; 1应用Real-time PCR方法显示,CCD后TRPV1的mRNA表达增加并应用免疫组化方法显示CCD后TRPV1表达增加,用Western blot的方法对CCD、CCI以及SNL三种神经损伤模型上CCR2在DRG中的表达进行了定量分析,结果表明,三种损伤方式都能引起CCR2的表达量增加,尤以CCD损伤后CCR2增加特别明显,在DRG组织切片和急性分离的DRG神经元上用免疫荧光方法显示CCR2和TRPV1共表达于CCD神经元,这就为MCP-1对TRPV1的调控作用提供形态学基础。 2动物自发痛反应行为测试结果显示,MCP-1增强Capsaicin诱发的疼痛行为,与单独给与MCP-1和Capsaicin有显著性差异。 3全细胞膜片钳记录显示,MCP-1增强在急性分离的CCD神经元上TRPV1受体激动剂Capsaicin诱导的膜电位去极化幅度及内向电流幅度。应用Ca~(2+)成像技术观察对急性分离的CCD神经元在MCP-1作用前后对Capsaicin诱发的细胞内游离钙离子的反应。结果表明,MCP-1(100 ng/ml)增强Capsaicin诱发的细胞内钙荧光强度 4在急性分离的CCD神经元上,应用Ca~(2+)成像技术观察PLC阻断剂U73122对MCP-1增强Capsaicin诱发的细胞内钙荧光强度的调控作用。结果显示U73122有效地阻断MCP-1对TRPV1的增强效应。动物自发痛反应行为测试结果显示,PLC阻断剂U73122有效地阻断MCP-1对Capsaicin引发的自发痛的易化作用。综上所述,提示CCD损伤后DRG神经元上调表达的CCR2,在MCP-1作用激活后可通过增强TRPV1的功能,使痛觉神经元的兴奋性提高,从而参与痛觉过敏的产生。
[Abstract]:Monocyte chemoattractant protein-1 (MCP-1) is a CC chemokine. Recent studies have shown that in the presence of neuropathic pain, inflammation or other pathological changes, the expression of chemokine MCP-1 and its receptor CCR2 is up-regulated in neurons and glial cells, and can directly induce a sustained increase in the excitability of pain neurons. Changes in gene expression of nociceptors. MCP-1 is considered to be a new member of the pain family and an important factor leading to pain. Capsaicin receptor TRPV1 is highly expressed in pain receptors such as class C unmyelinated fibers or A 未 nerve fibers in small and medium-sized neurons such as dorsal root ganglion, trigeminal ganglion and so on. It is a ligand gated non-selective cationic channel. More and more studies have shown that TRPV1 plays an important role in the generation of pain and the enhancement of pain sensitivity. In order to further study the mechanism of MCP-1 regulating the pain effect of TRPV1, the model of chronic compression of dorsal root ganglion (DRG) in rats was studied. The expression and distribution of MCP-1 receptor CCR2 and TRPV1 on CCD dorsal root ganglion cells were determined by Real-time PCR and immunohistochemical staining, and the expression of CCR2 in DRG was quantitatively analyzed by Western blot method. Behavioral test was used to observe whether MCP-1 facilitated the pain induced by TRPV1 on CCD rats. Whole cell patch-clamp and calcium imaging were used. The effect of MCP-1 on TRPV1 channel in DRG neurons of CCD rats and the possible intracellular signal pathway were observed. The main results are as follows; 1 the expression of mRNA in TRPV1 was increased by Real-time PCR method and the expression of TRPV1 in CCD was detected by immunohistochemistry. The expression of CCR2 in DRG was quantitatively analyzed by Western blot method. The expression of CCR2 was increased in all three kinds of injury modes, especially CCR2 after CCD injury. CCR2 and TRPV1 co-expressed in CCD neurons by immunofluorescence method in DRG tissue sections and acute isolated DRG neurons. This provides a morphological basis for the regulation of TRPV1 by MCP-1. 2 the results of spontaneous pain response test showed that MCP-1 enhanced the pain behavior induced by Capsaicin, which was significantly different from that of MCP-1 and Capsaicin alone. 3 whole cell patch clamp records showed that MCP-1 enhanced the depolarization amplitude and inward current amplitude induced by TRPV1 receptor agonist Capsaicin on the acutely isolated CCD neurons. Ca~(2 imaging was used to observe the response of acutely isolated CCD neurons to intracellular free calcium ions induced by Capsaicin before and after MCP-1 treatment. The results showed that MCP-1 + 100ng / ml enhanced intracellular calcium fluorescence induced by Capsaicin. 4 in acute isolated CCD neurons, the effect of U73122, a PLC blocker, on the intracellular calcium fluorescence intensity induced by Capsaicin induced by MCP-1 was observed by Ca~(2 imaging technique. The results showed that U73122 effectively blocked the enhancement effect of MCP-1 on TRPV1. The results of animal spontaneous pain response test showed that U73122 could effectively block the facilitation of Capsaicin induced spontaneous pain by MCP-1. In conclusion, it is suggested that the up-regulated expression of CCR2 in DRG neurons after CCD injury can increase the excitability of pain neurons by enhancing the function of TRPV1 after the activation of MCP-1, and thus participate in the production of hyperalgesia.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

【引证文献】