本文關(guān)鍵詞： 軸突轉(zhuǎn)運(yùn) 背根神經(jīng)節(jié) GIRK通道 神經(jīng)損傷 神經(jīng)病理性疼痛 脊髓　出處：《哈爾濱工業(yè)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：傷害性神經(jīng)元興奮性的增強(qiáng)是慢性疼痛發(fā)生和發(fā)展的基礎(chǔ)。多種離子通道,包括鈉離子通道、鈣離子通道和鉀離子通道在控制神經(jīng)元的興奮性上起到重要作用。G蛋白門控內(nèi)向整流鉀離子(GIRK)通道已被證實(shí)能夠調(diào)控神經(jīng)元的興奮性。此前的研究報(bào)道GIRK通道四個(gè)亞單位的m RNA在大鼠和人類背根神經(jīng)節(jié)(DRG)中均有所表達(dá)。功能性的GIRK通道是由其亞單位成員中的一個(gè)或兩個(gè)組成的同源四聚體或異源四聚體。GIRK通道通過與抑制性G蛋白偶聯(lián)受體(G protein coupled receptors,GPCRs)的相互作用而被激活并開放,鉀離子外流,使膜電位變得更“負(fù)”,從而降低神經(jīng)元的興奮性。本論文旨在系統(tǒng)地研究GIRK通道亞單位在正常大鼠DRG和脊髓中表達(dá)的神經(jīng)化學(xué)特性,以及通過構(gòu)建外周神經(jīng)損傷的大鼠神經(jīng)病理性疼痛模型研究外周神經(jīng)損傷對(duì)GIRK通道四個(gè)亞單位的表達(dá)調(diào)控,分析和闡述GIRK通道在神經(jīng)病理性疼痛發(fā)生和發(fā)展中所起到的作用。本研究利用免疫組織化學(xué)實(shí)驗(yàn)和定量分析等方法確定了GIRK通道亞單位在正常大鼠腰椎4-5(L4-5)節(jié)段DRG組織和L5脊髓組織中的表達(dá)和分布情況。在正常大鼠DRG中~70%和10%的神經(jīng)元分別表達(dá)GIRK1和GIRK2。利用降鈣素基因相關(guān)肽(CGRP)、植物凝集素B4(IB4)和神經(jīng)絲蛋白-200(NF200)抗體分別標(biāo)記DRG中的小型無髓鞘肽能神經(jīng)元、小型無髓鞘非肽能神經(jīng)元和中大型有髓鞘神經(jīng)元,激光共聚焦共定位研究結(jié)果表明GIRK1和GIRK3廣泛地表達(dá)于這三種神經(jīng)元類群,而GIRK2僅表達(dá)于CGRP和NF200陽(yáng)性神經(jīng)元類群。進(jìn)一步使用四種鈣離子結(jié)合蛋白鈣結(jié)合蛋白D28k(CB)、鈣視網(wǎng)膜蛋白(CR)、小清蛋白(PV)和促泌素蛋白(Scgn)抗體標(biāo)記不同的DRG神經(jīng)元類群,對(duì)GIRK通道亞單位的表達(dá)特性進(jìn)行進(jìn)行地分析,結(jié)果表明GIRK1與四種鈣離子結(jié)合蛋白均存在不同程度上的共定位關(guān)系,而GIRK2只在CB陽(yáng)性神經(jīng)元中表達(dá)。為分析GIRK1~4在脊髓背角中的表達(dá)特性,使用不同的標(biāo)記物分別標(biāo)記DRG神經(jīng)元軸突在脊髓背角投射末端分布的薄層(lamina),其中,GIRK1和GIRK2主要表達(dá)于lamina II,GIRK3和GIRK4主要表達(dá)于lamina I和外層lamina II。此外,GIRK1~4與VGLUT1或VGLUT2的共定位研究結(jié)果表明GIRK1~4在脊髓背角存在突觸前膜定位。利用神經(jīng)元標(biāo)記物Neu N標(biāo)記脊髓背角神經(jīng)元,結(jié)果表明GIRK1和GIRK2廣泛地表達(dá)于不同lamina區(qū)域的局部神經(jīng)元,但是GIRK3和GIRK4不表達(dá)于局部神經(jīng)元。為研究外周神經(jīng)損傷對(duì)GIRK通道亞單位表達(dá)調(diào)控,我們通過大鼠坐骨神經(jīng)離斷(axotomy)手術(shù)制備了外周神經(jīng)損傷引起的神經(jīng)病理性疼痛模型。通過免疫組織化學(xué)實(shí)驗(yàn)、原位雜交實(shí)驗(yàn)、實(shí)時(shí)熒光定量PCR實(shí)驗(yàn)、western blot實(shí)驗(yàn)和定量分析等方法對(duì)GIRK1~4在mRNA和蛋白質(zhì)水平上的表達(dá)情況進(jìn)行分析。結(jié)果表明損傷側(cè)DRG和脊髓背角中GIRK1、GIRK2和GIRK4的表達(dá)均顯著下調(diào),而GIRK3的表達(dá)顯著上調(diào)。結(jié)合此前的研究結(jié)果進(jìn)行分析,GIRK3表達(dá)的上調(diào)能夠降低神經(jīng)元細(xì)胞膜的GIRK1/2和GIRK2/2通道。為研究GIRK通道亞單位在DRG神經(jīng)元中的軸突轉(zhuǎn)運(yùn),分別利用大鼠坐骨神經(jīng)結(jié)扎(ligation)模型和脊髓神經(jīng)背根切除(rhizotomy)模型研究在DRG神經(jīng)元中合成的GIRK1~4向外周端和中樞端運(yùn)輸情況。實(shí)驗(yàn)結(jié)果表明GIRK1~4均存在向外周和中樞運(yùn)輸?shù)捻樞修D(zhuǎn)運(yùn),此外,GIRK1~3存在外周軸突末端向胞體端運(yùn)輸?shù)哪嫘修D(zhuǎn)運(yùn)。利用免疫組織化學(xué)實(shí)驗(yàn),進(jìn)一步檢測(cè)了GIRK1~4在正常大鼠坐骨神經(jīng)和后爪無毛皮膚中的表達(dá)和分布,結(jié)果表明GIRK1~4廣泛地表達(dá)于包括感覺纖維在內(nèi)的多種類群的神經(jīng)纖維,且GIRK1~4均表達(dá)于后爪無毛皮膚真皮層中的神經(jīng)末端。綜上所述,本研究首次系統(tǒng)地闡述了GIRK通道四種亞單位在大鼠DRG和脊髓中表達(dá)的神經(jīng)化學(xué)特性,并通過外周神經(jīng)損傷模型和多種分子生物學(xué)實(shí)驗(yàn)方法證明了GIRK通道在神經(jīng)病理性疼痛情況下的表達(dá)下調(diào)。GIRK通道表達(dá)的下調(diào)會(huì)引起感覺神經(jīng)元的過度興奮,可能是神經(jīng)病理性疼痛產(chǎn)生和長(zhǎng)時(shí)程維持的重要原因。
[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.

【學(xué)位授予單位】：哈爾濱工業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R741

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