外周神经损伤对DRG和脊髓神经细胞GIRK家族蛋白表达的影响

发布时间：2018-02-13 12:54

本文关键词： 轴突转运背根神经节 GIRK通道神经损伤神经病理性疼痛脊髓　出处：《哈尔滨工业大学》2016年博士论文　论文类型：学位论文

【摘要】：伤害性神经元兴奋性的增强是慢性疼痛发生和发展的基础。多种离子通道,包括钠离子通道、钙离子通道和钾离子通道在控制神经元的兴奋性上起到重要作用。G蛋白门控内向整流钾离子(GIRK)通道已被证实能够调控神经元的兴奋性。此前的研究报道GIRK通道四个亚单位的m RNA在大鼠和人类背根神经节(DRG)中均有所表达。功能性的GIRK通道是由其亚单位成员中的一个或两个组成的同源四聚体或异源四聚体。GIRK通道通过与抑制性G蛋白偶联受体(G protein coupled receptors,GPCRs)的相互作用而被激活并开放,钾离子外流,使膜电位变得更“负”,从而降低神经元的兴奋性。本论文旨在系统地研究GIRK通道亚单位在正常大鼠DRG和脊髓中表达的神经化学特性,以及通过构建外周神经损伤的大鼠神经病理性疼痛模型研究外周神经损伤对GIRK通道四个亚单位的表达调控,分析和阐述GIRK通道在神经病理性疼痛发生和发展中所起到的作用。本研究利用免疫组织化学实验和定量分析等方法确定了GIRK通道亚单位在正常大鼠腰椎4-5(L4-5)节段DRG组织和L5脊髓组织中的表达和分布情况。在正常大鼠DRG中~70%和10%的神经元分别表达GIRK1和GIRK2。利用降钙素基因相关肽(CGRP)、植物凝集素B4(IB4)和神经丝蛋白-200(NF200)抗体分别标记DRG中的小型无髓鞘肽能神经元、小型无髓鞘非肽能神经元和中大型有髓鞘神经元,激光共聚焦共定位研究结果表明GIRK1和GIRK3广泛地表达于这三种神经元类群,而GIRK2仅表达于CGRP和NF200阳性神经元类群。进一步使用四种钙离子结合蛋白钙结合蛋白D28k(CB)、钙视网膜蛋白(CR)、小清蛋白(PV)和促泌素蛋白(Scgn)抗体标记不同的DRG神经元类群,对GIRK通道亚单位的表达特性进行进行地分析,结果表明GIRK1与四种钙离子结合蛋白均存在不同程度上的共定位关系,而GIRK2只在CB阳性神经元中表达。为分析GIRK1~4在脊髓背角中的表达特性,使用不同的标记物分别标记DRG神经元轴突在脊髓背角投射末端分布的薄层(lamina),其中,GIRK1和GIRK2主要表达于lamina II,GIRK3和GIRK4主要表达于lamina I和外层lamina II。此外,GIRK1~4与VGLUT1或VGLUT2的共定位研究结果表明GIRK1~4在脊髓背角存在突触前膜定位。利用神经元标记物Neu N标记脊髓背角神经元,结果表明GIRK1和GIRK2广泛地表达于不同lamina区域的局部神经元,但是GIRK3和GIRK4不表达于局部神经元。为研究外周神经损伤对GIRK通道亚单位表达调控,我们通过大鼠坐骨神经离断(axotomy)手术制备了外周神经损伤引起的神经病理性疼痛模型。通过免疫组织化学实验、原位杂交实验、实时荧光定量PCR实验、western blot实验和定量分析等方法对GIRK1~4在mRNA和蛋白质水平上的表达情况进行分析。结果表明损伤侧DRG和脊髓背角中GIRK1、GIRK2和GIRK4的表达均显著下调,而GIRK3的表达显著上调。结合此前的研究结果进行分析,GIRK3表达的上调能够降低神经元细胞膜的GIRK1/2和GIRK2/2通道。为研究GIRK通道亚单位在DRG神经元中的轴突转运,分别利用大鼠坐骨神经结扎(ligation)模型和脊髓神经背根切除(rhizotomy)模型研究在DRG神经元中合成的GIRK1~4向外周端和中枢端运输情况。实验结果表明GIRK1~4均存在向外周和中枢运输的顺行转运,此外,GIRK1~3存在外周轴突末端向胞体端运输的逆行转运。利用免疫组织化学实验,进一步检测了GIRK1~4在正常大鼠坐骨神经和后爪无毛皮肤中的表达和分布,结果表明GIRK1~4广泛地表达于包括感觉纤维在内的多种类群的神经纤维,且GIRK1~4均表达于后爪无毛皮肤真皮层中的神经末端。综上所述,本研究首次系统地阐述了GIRK通道四种亚单位在大鼠DRG和脊髓中表达的神经化学特性,并通过外周神经损伤模型和多种分子生物学实验方法证明了GIRK通道在神经病理性疼痛情况下的表达下调。GIRK通道表达的下调会引起感觉神经元的过度兴奋,可能是神经病理性疼痛产生和长时程维持的重要原因。
[Abstract]:Damage increased excitability of neurons is the basis of the occurrence and development of chronic pain. A variety of ion channels, including sodium channels, calcium channels and potassium channels play an important role in.G protein gated inward rectifier potassium in controlling neuronal excitability on ion channel (GIRK) has been shown to modulate excitability of neurons after it was reported that GIRK Channel Four subunit m RNA in rat and human dorsal root ganglion (DRG) were expressed. GIRK functional channels by its subunit members in one or two of four dimers consisting of homologous or heterologous four tetrameric.GIRK channel with inhibition G protein coupled receptors (G protein coupled receptors, GPCRs) interactions are activated and open, the efflux of potassium ions, the membrane potential becomes more negative, thereby reducing the excitability of the neuron. This paper aims to systematically study GIRK Neurochemical features of expression of channel subunits in DRG and spinal cord in normal rats, and through the construction of peripheral neuropathic pain rat model of nerve injury of peripheral nerve injury on the expression regulation of GIRK Channel Four subunit, analysis and elaboration of GIRK channel plays a role in the development of neuropathy and neuropathic pain. Immunohistochemistry and quantitative analysis using this research method to determine the GIRK channel subunits in normal rat lumbar 4-5 (L4-5) expression and distribution of segmental DRG tissue and L5 in spinal cord. ~70% in the normal rat DRG and 10% neurons respectively the expression of GIRK1 and GIRK2. by calcitonin gene related peptide (CGRP), lectin B4 (IB4) and neurofilament protein -200 (NF200) antibodies were labeled with DRG in small unmyelinated peptidergic neurons, non small unmyelinated peptidergic neurons and large myelinated Neurons, confocal colocalization results showed that GIRK1 and GIRK3 are widely expressed in these three groups of neurons, while GIRK2 was only expressed in CGRP and NF200 positive neurons in groups. Further use of four calcium binding protein calcium binding protein D28k (CB), calcium (CR), retinal protein parvalbumin (PV) and secretagogue protein (Scgn) antibody labeled DRG neurons in different groups, carried out analysis on the expression characteristics of GIRK channel subunits, the results showed that GIRK1 and four calcium binding proteins are Co located in different degree, and GIRK2 only expressed in CB positive neurons. The expression for characteristic analysis GIRK1~4 in spinal dorsal horn, using different markers were labeled with DRG thin axons in the spinal dorsal horn projection terminal distribution (lamina), wherein, GIRK1 and GIRK2 mainly expressed on lamina II, GIRK3 and GIRK4 were mainly expressed in the Lamina I and lamina II. in GIRK1~4 and the outer layer, VGLUT1 or VGLUT2 co localization results showed that GIRK1~4 existed in the spinal dorsal horn presynaptic localization. The neuron marker Neu N labeled neurons in the spinal dorsal horn neurons, the results show that local GIRK1 and GIRK2 are widely expressed in different lamina regions, but GIRK3 and GIRK4 expression in the local neurons. For the study of peripheral nerve injury on GIRK channel subunit expression regulation, we isolated the rat sciatic nerve (axotomy) surgery preparation model of neuropathic pain induced by peripheral nerve injury. By immunohistochemistry, in situ hybridization, real-time PCR experiment, Western experiment and quantitative blot analysis method to analyze the expression of GIRK1~4 at mRNA and protein levels. The results showed that the damage of GIRK1 side DRG and dorsal horn of the spinal cord, GIRK2 and GIRK4 The expression was significantly reduced, while the GIRK3 expression increased significantly. Combined with previous research results, the up regulation of GIRK3 expression can reduce the neuronal membrane GIRK1/2 and GIRK2/2 channels. For the study of GIRK channel subunits of axonal transport in DRG neurons, respectively by sciatic nerve ligation in rats (ligation) and spinal cord dorsal nerve model root excision (rhizotomy) model to study the synthesis of GIRK1~4 in DRG neurons to peripheral and central endings of transport. The experimental results show that GIRK1~4 has peripheral and central transport of anterograde transport, in addition, the presence of GIRK1~3 peripheral axon terminal to retrograde transport transport. The end cell immunohistochemical experiments further examined the expression and distribution of GIRK1~4 in the skin of hairless claw normal rat sciatic nerve and the results show that GIRK1~4 is widely expressed in many types of sensory fibers, including Group of nerve fibers, and GIRK1~4 were expressed in nerve endings in the dermis to claw glabrous skin. In summary, this study first systematically expounded the neurochemical features of the expression of GIRK Channel Four subunit in DRG and spinal cord in rats, and the model of peripheral nerve injury and multiple kinds of molecular biology experiments proved that the expression of GIRK channels in neuropathic pain under the down-regulation of.GIRK expression channel can cause sensory neuronal hyperexcitability, may be an important cause of neuropathic pain generation and maintenance of long-term.

【学位授予单位】：哈尔滨工业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R741

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