大鼠背根节和脊髓内ClC-3参与神经病理性痛信息调控的研究

发布时间：2018-01-09 03:28

本文关键词：大鼠背根节和脊髓内ClC-3参与神经病理性痛信息调控的研究　出处：《第四军医大学》2012年硕士论文　论文类型：学位论文

【摘要】：电压门控性氯离子通道3型（voltage-gated chloride channel type3,ClC-3）在体内的分布十分广泛，对它的研究比较深入和明确，已知它与多种生理功能的调节有关，如细胞容积调节、细胞酸化、肿瘤转移、癫痫等。体内的ClC-3主要分布在心肌、支气管上皮、视网膜及一些肿瘤细胞，在神经系统主要分布在突触部位。氯离子通道的激活有抑痛作用，鞘内给予氯离子通道激动剂GABA对各种原因导致的慢性痛均有显著的镇痛效应。 5-硝基-2,3-（苯丙胺）-苯甲酸（NPPB）是一种具有较高选择性和效能的氯离子通道阻滞剂，对动物和培养细胞都有较明显的作用，，被广泛的应用于氯离子通道的相关研究。神经损伤所导致的神经病理性痛是临床常见的慢性痛的病因，因其长期迁延不愈而给患者带来了巨大的痛苦。目前对神经病理性痛缺乏有效的治疗手段，因此探索其发病机制有助于寻找治疗靶点，解除患者病痛。离子通道的激活和下调在神经病理性痛中起着重要的作用，既往研究主要集中在阳离子通道，如Nav1.7、Nav1.8、Cav1.2等，而对阴离子通道的研究较少。氯离子是体内最丰富的阴离子，在调节神经系统兴奋性方面具有重要作用。ClC-3是调节神经元内氯离子浓度的重要离子通道，但是ClC-3在神经病理性痛中的作用尚一无所知。本研究应用腓总神经（commonperonealnerve, CPN）结扎导致神经病理性痛模型，结合氯离子通道阻滞剂NPPB，用行为学及免疫组织化学方法观察ClC-3在神经病理性痛中的作用，旨在为临床镇痛提供新的靶点。一、ClC-3在腓总神经结扎神经病理性痛模型大鼠脊髓背角和背根神经节的表达变化用免疫组织化学染色结果表明，ClC-3主要分布在正常大鼠脊髓背角浅层，特别是Ⅰ层；在DRG，ClC-3分布在神经元的胞膜上。腓总神经结扎后7d，ClC-3在DRG和脊髓背角表达上调，随着病情的发展，ClC-3表达逐渐下降，以上结果利用Westernblot得到证实。提示ClC-3在腓总神经结扎所致的神经病理性痛的不同时相起着不同的作用。二、阻断ClC-3对痛行为的影响在CPN结扎大鼠，鞘内给予氯离子通道阻滞剂NPPB5μl，利用vonFrey丝和Hargreaves法检测大鼠的机械缩足反射阈值（MWT）和热缩足反射期（TWL），观察到NPPB+CPN结扎组大鼠的MWT和TWL低于鞘内给予DMSO的CPN结扎大鼠，且该效应在CPN结扎7d时最为明显。以上结果表明，ClC-3较为特异性的分布在正常大鼠的DRG神经元和脊髓背角浅层；在CPN结扎导致神经病理性痛模型大鼠，ClC-3在DRG和脊髓背角的表达早期上调，晚期逐渐降低；鞘内给予NPPB阻断ClC-3可使CPN结扎大鼠的机械痛阈和热痛阈下降。以上结果从形态学和行为学方面提示ClC-3可能参与神经病理性痛信息的传递和调控。
[Abstract]:The voltage-gated chloride channel 3 (voltage-gated chloride channel type3, ClC-3) is widely distributed in the body, the study on it deeply and clearly, it is known with a variety of physiological functions such as regulation, cell volume regulation, intracellular acidification, tumor metastasis, such as epilepsy. In vivo ClC-3 mainly distributed in the myocardium. The bronchial epithelium, retina and some tumor cells in the nervous system are mainly distributed in the synapses. Activated chloride channel inhibition have analgesic effect, analgesic effect of intrathecal chloride channel agonist GABA of all causes were slow pain significantly.
5- nitro -2,3- (amphetamine) benzoic acid (NPPB) is a highly selective and effective chloride channel blocker. It plays a more significant role in animal and cultured cells, and is widely applied in the study of chloride channels.
Neuropathic pain caused by nerve injury is a common clinical cause of chronic pain, because of the long-term unhealed patients caused great pain of neuropathic pain. At present there is no effective treatment, so it is helpful to find therapeutic targets and explore its pathogenesis, relieve pain in patients. Reduction plays an important role in neuropathic pain and the activation of ion channels, previous studies mainly focused on ion channels, such as Nav1.7, Nav1.8, Cav1.2, and the research on the anion channel is less. The chloride ion is the most abundant anions in vivo, in the regulation of neuronal excitability plays an important role in.ClC-3 is an important regulation of ion channels of chloride ion concentration in neurons however, the role of ClC-3 in neuropathic pain. This study is absolutely ignorant of common peroneal nerve (commonperonealnerve, CPN) ligation can cause nerve pathology The role of ClC-3 in neuropathic pain was observed by behavioral and immunohistochemical methods combined with chloride channel blocker NPPB.
The expression of ClC-3 in the dorsal horn of spinal cord and dorsal root ganglion in the rat model of the neuropathic pain model of the peroneal nerve ligation
Immunohistochemical staining results showed that ClC-3 mainly distributed in the normal rat spinal cord dorsal horn, especially the 1 layer; in DRG, the distribution of ClC-3 in the membrane of neurons. The common peroneal nerve after ligation of 7D, DRG and ClC-3 expression in the dorsal horn of the spinal cord, as the disease progresses, the expression of ClC-3 decreased gradually the above results are confirmed by Westernblot, suggest that ClC-3 in neuropathic pain caused by ligation of the common peroneal nerve at different phases plays a different role.
Two, the effect of blocking ClC-3 on pain behavior
In CPN rats, intrathecal administration of chloride channel blocker NPPB5 L, using vonFrey wire and Hargreaves method to detect the rat mechanical withdrawal threshold (MWT) and thermal withdrawal period (TWL), observed NPPB+CPN ligated rats MWT and TWL lower than that of CPN rats with intrathecal injection of DMSO the effect on CPN, and ligation of the 7d was the most obvious.
The above results show that the distribution of ClC-3 is more specific in the normal rat DRG neurons and the superficial layers of the spinal dorsal horn; leading to neuropathic pain model rats after CPN ligation, ClC-3 in DRG and spinal dorsal horn expression in early rise, late gradually decreased; intrathecal NPPB blockade of ClC-3 can make the mechanical pain threshold and heat pain threshold CPN ligation of rats decreased. The above results from morphological and behavioral studies suggest that ClC-3 might be involved in neuropathic pain information transmission and regulation.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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