DRG局部GABA系统在神经病理性疼痛中的作用

发布时间：2018-06-29 09:16

本文选题：背根神经节 + GABA回路　；参考：《河北医科大学》2015年硕士论文

【摘要】：躯体感觉神经元是机体感觉系统的重要组成部分,其胞体存在于背根神经节(dorsal root ganglion,DRG),DRG神经元外周轴突末梢可感受各种各样的外周刺激例如温度刺激、化学刺激和机械刺激等,并将其转化为神经冲动向中枢传递,从而形成温度觉、痛觉和触觉等感觉。我们前期的研究发现DRG神经元不仅具有痛觉信号形成和传递作用,而且还兼具痛觉信号调节作用。DRG局部存在功能性的GABA回路系统,DRG组织和培养细胞在多种兴奋性刺激作用下可有GABA释放,DRG局部应用GABA对缓激肽诱发的急性炎性疼痛有显著的抑制作用。这些均提示DRG部位(特别是该部位的GABA系统)可能作为疼痛治疗的潜在靶点。慢性神经病理性疼痛是临床常见疾病,其发病机制尚不清楚,目前临床针对慢性神经病理性疼痛尚缺乏有效的治疗手段。本论文中我们将重点关注DRG局部GABA系统在慢性神经病理性疼痛的发生和治疗中的作用。首先,为了研究DRG局部GABA系统在疼痛发生过程中的作用,我们建立了一个全新的“sniffer patch”电生理实验方法,证明大鼠DRG神经元在兴奋情况下可直接释放GABA,这为DRG局部存在功能性的GABA系统提供了更直接的证据。其次,我们通过制备大鼠坐骨神经慢性挤压损伤(CCI)神经病理性疼痛模型,观察疼痛进展期DRG神经元GABA电流密度的改变,以研究DRG局部GABA系统在神经病理性疼痛发生中的作用。最后,我们通过渗透泵靶向DRG给药系统,观察GABA对神经性病理性疼痛反应的影响,分析DRG局部GABA系统作为慢性神经病理性疼痛治疗靶点的可能性。第一部分通过“Sniffer Patch”方法证明DRG神经元的GABA释放功能目的:建立一种全新的电生理实验方法“sniffer patch”,证明DRG神经元具有直接释放GABA的功能。方法:打孔“sniffer patch”电生理实验方法,半定量PCR技术VGAT-ChR2-YFP小鼠鉴定,冰冻切片-免疫荧光技术,统计方法为t检验。结果:①药物刺激DRG神经元“Sniffer Patch”结果:将表达有GABAA受体的HEK293B细胞与大鼠急性分离DRG细胞共培养,与小直径DRG紧密相邻的转染了 GABAA受体的HEK293B细胞在200μLM GABA作用下产生很大的GABA-Cl-电流。而当给予TRPV1受体的激动剂辣椒素激活小直经DRG神经元,用sniffer patch的方法在相邻HEK293B细胞记录到类似的GABA样电流。而辣椒素不能诱发单独培养的转染了 GABAA受体的HEK293B细胞,以及与DRG共培养的空白HEK293B细胞产生任何电流。②机械刺激DRG神经元的“Sniffer Patch”结果:GABAA受体转染HEK293B细胞与大鼠DRG细胞共培养,给予表达有GAB AA受体的HEK293B细胞紧密相邻的小直径DRG机械刺激,未能诱发HEK293B细胞产生GABA样电流。③光激活VGAT-ChR2-YFP 小鼠 DRG 神经元的“Sniffer Patch”结果:分离VGAT-ChR2-YFP小鼠DRG细胞,如上述方法进行共培养,在473nm激光刺激下,在与转基因小鼠VGAT-ChR2-YFP的DRG紧密相邻的表达有GABAA受体的HEK293B细胞记录到一个小的内向电流,比在单独培养的表达有GABAA受体的HEK293B细胞,以及与野生型小鼠的DRG紧密相邻的表达有GABAA受体的HEK293B细胞记录的电流大,且具有统计学意义。结论:大鼠DRG神经元在capsaicin刺激下可直接释放GABA,转基因小鼠VGAT-ChR2-YFP的DRG神经元在473 nm激光刺激下也有类似GABA释放现象,从而证实了 DRG神经元具有直接释放GABA的功能。为DRG局部存在功能性的GABA系统提供了更直接的证据。第二部分DRG部位GABA系统对神经病理性疼痛的影响目的:研究CCI手术后不同直径大小DRG神经元的GABA电流密度的改变。采用渗透泵靶向DRG给药的给药方法,观察DRG局部GABA系统对病理性疼痛的治疗作用。方法:制备神经病理性模型CCI;应用Von Frey纤维刺痛针和热刺痛仪,测定大鼠机械痛、热痛阈值;建立渗透泵靶向DRG给药系统;全细胞膜片钳技术;统计方法为两个独立样本的非参数检验Mann-Whitney Test。结果:①CCI模型建立:CCI组出现明显的痛觉过敏症状,其机械痛和热痛阈值在术后第一天明显下降,并持续至第十四天,与对照组比存在显著性差异(P0.05)。②电生理结果:用电生理技术分别记录大鼠CCI手术后第五天和第十四天的术侧和对侧大鼠大、中、小直径L4-6DRG神经元的GABA-Cl-电流。大直径和小直径L4-6DRG神经元CCI术后第五天、第十四天术侧DRG神经元的电流密度和对GABA的反应数目与对侧DRG神经元无明显区别。中等直径L4-6DRG神经元在CCI术后第五天大鼠术侧的DRG神经元GABA-C1-电流密度大于对侧(P0.05),而第十四天术侧与对侧无显著区别。第五天和第十四天对侧的DRG神经元对GABA有反应的神经元数目远少于术侧。③行为学实验结果:通过渗透泵靶向DRG给药系统对CCI模型大鼠部位持续给予GABA(200μM)个周,对照组给予生理盐水;在术前及术后第1,5,7,10,14天进行机械痛和热痛实验的测定。结果显示GABA的应用可使术后第一和第五天热痛阈值明显增大(P0.05),而GABA的应用对CCI大鼠机械痛阈无明显影响。结论:以上实验证明在神经病理性疼痛发生中伴有一定DRG局部GABA系统敏感性变化,特别是中直径DRG神经元GABA敏感性增高更为明显;在神经病理性疼痛发生过程中激活DRG局部GABA系统可一定程度上缓解疼痛症状,尤其是对热痛过敏症状的缓解作用更为突出。
[Abstract]:Somatosensory neurons are important components of the sensory system of the body, and their bodies exist in the dorsal root ganglion (dorsal root ganglion, DRG). The peripheral axons of DRG neurons can feel a variety of peripheral stimuli such as temperature stimulation, chemical stimulation and mechanical stimulation, and transform them into nerve impulses to the center, thus forming a nerve impulse. Our previous studies have found that DRG neurons not only have the formation and transmission of pain signals, but also have a functional GABA loop system in which.DRG is localized in the regulation of pain signals, and the DRG tissue and culture cells can be released by GABA under a variety of excitatory stimuli, and DRG local application of GABA. The acute inflammatory pain induced by bradykinin has a significant inhibitory effect. These all suggest that the DRG site (especially the GABA system in this area) may be a potential target for pain treatment. Chronic neuropathic pain is a common clinical disease, its pathogenesis is not yet clear, and there is still a lack of effective treatment for chronic neuropathic pain at present. In this paper, we will focus on the role of DRG local GABA system in the occurrence and treatment of chronic neuropathic pain. First, in order to study the role of DRG local GABA system in the process of pain, we have established a new "sniffer patch" electrophysiological experiment to prove that the rat DRG neurons are in the process. GABA can be released directly under the excitation condition, which provides more direct evidence for the functional GABA system in the local DRG. Secondly, we observe the GABA current density of DRG neurons in the DRG neurons of the pain progression by preparing the neuropathic pain model of the chronic crush injury (CCI) of the rat sciatic nerve, to study the neuropathy in the DRG local GABA system in the neuropathy. The role of rational pain in the occurrence of pain. Finally, we observe the effect of GABA on neuropathic pain by targeting the osmotic pump targeting DRG drug delivery system, and analyze the possibility of DRG local GABA system as a target for chronic neuropathic pain treatment. The first part uses the "Sniffer Patch" method to prove the GABA release function of DRG neurons. Objective: to establish a new method of electrophysiological experiment "sniffer patch", which proves that DRG neurons have the function of releasing GABA directly. Method: electrophysiological experimental method of "sniffer patch", semi quantitative PCR technique, VGAT-ChR2-YFP mouse identification, frozen section immunofluorescence technique, and statistical method for t test. Results: (1) drug stimulates DRG. The result of neuron "Sniffer Patch": co culture the HEK293B cells expressing the GABAA receptor with the acute isolated DRG cells of rats. The HEK293B cells transfected with the GABAA receptor, which is closely adjacent to the small diameter DRG, produce a large GABA-Cl- current under the action of 200 micron GABA. Neurons, sniffer patch method was used to record similar GABA like current in adjacent HEK293B cells. Capsaicin could not induce HEK293B cells transfected with GABAA receptor alone, and the blank HEK293B cells co cultured with DRG produce any current. 2. The "Sniffer Patch" result of the DRG nerve element stimulation: GABAA receptor turns. HEK293B cells were co cultured with rat DRG cells, and the HEK293B cells expressing the GAB AA receptor were closely adjacent to the small diameter DRG mechanical stimulation and failed to induce the HEK293B cells to produce the GABA like current. Co culture, under 473nm laser stimulation, a small inward current was recorded by HEK293B cells expressing GABAA receptor with the GABAA receptor closely adjacent to the DRG of the transgenic mouse DRG, and the HEK293B cells expressing GABAA receptors in the isolated culture, and HEK293B with the GABAA receptor in close proximity to the DRG of the wild type mice. The cell recording current is large and has statistical significance. Conclusion: the rat DRG neurons can release GABA directly under the stimulation of capsaicin. The DRG neurons of the transgenic mice VGAT-ChR2-YFP also have the similar GABA release under the 473 nm laser stimulation, which confirms that the DRG neurons have the function of releasing GABA directly. It has the function of the DRG part. The GABA system provides more direct evidence. Second part of the effect of the DRG site GABA system on neuropathic pain is to study the changes in the GABA current density of DRG neurons of different diameters in CCI after CCI operation. The therapeutic effect of the local GABA system on the pathological pain of DRG is observed by using the osmotic pump target to the DRG administration. Method: the neuropathic model CCI was prepared; the mechanical pain and the heat pain threshold of the rats were measured with the Von Frey fiber prick needle and the thermo pain instrument; the osmotic pump targeted DRG administration system was established; the whole cell patch clamp technique was established; the statistical method was the non parametric test of the Mann-Whitney Test. results of two independent samples: (1) the CCI model was established: the CCI group appeared obvious pain. The threshold of mechanical pain and heat pain decreased significantly on the first day after the operation and lasted to fourteenth days, and there was a significant difference compared with the control group (P0.05). 2. Electrophysiological results: electrophysiological techniques were used to record the GABA-Cl- of the large, medium and small diameter L4-6DRG neurons in the fifth and fourteenth days of the rats after the operation and the fifth and fourteenth days after the operation. The current density of DRG neurons and the number of responses to GABA were not significantly different from that of the contralateral DRG neurons on the fourteenth day after the operation of the large diameter and small diameter L4-6DRG neurons on the fourteenth day. The GABA-C1- current density of the DRG neurons on the side of the middle diameter L4-6DRG neuron fifth days after CCI was greater than that of the contralateral DRG (P0.05), and Fourteenth There was no significant difference between the operation side and the contralateral side. The number of neurons that reacted to the contralateral DRG neurons on the fifth and fourteenth days was far less than that of the operation. (3) the results of the behavioral experiment: the CCI model rats were continuously given GABA (200 u M) weeks by the osmotic pump target to the CCI model rats, and the control group was given physiological saline; before and after the operation, 1,5,7 was given. The results of the test of mechanical pain and heat pain in 10,14 days showed that the application of GABA could significantly increase the heat pain threshold of the first and fifth days after operation (P0.05), and the application of GABA had no obvious effect on the mechanical pain threshold of CCI rats. In particular, the increased sensitivity of GABA in the middle diameter DRG neurons is more obvious. In the process of neuropathic pain, activation of DRG local GABA system can relieve pain symptoms to a certain extent, especially for the relief of heat pain allergic symptoms.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R402

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