本文關鍵詞： 軸突轉運 背根神經節(jié) GIRK通道 神經損傷 神經病理性疼痛 脊髓　出處：《哈爾濱工業(yè)大學》2016年博士論文　論文類型：學位論文

【摘要】：傷害性神經元興奮性的增強是慢性疼痛發(fā)生和發(fā)展的基礎。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元的興奮性上起到重要作用。G蛋白門控內向整流鉀離子(GIRK)通道已被證實能夠調控神經元的興奮性。此前的研究報道GIRK通道四個亞單位的m RNA在大鼠和人類背根神經節(jié)(DRG)中均有所表達。功能性的GIRK通道是由其亞單位成員中的一個或兩個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”,從而降低神經元的興奮性。本論文旨在系統(tǒng)地研究GIRK通道亞單位在正常大鼠DRG和脊髓中表達的神經化學特性,以及通過構建外周神經損傷的大鼠神經病理性疼痛模型研究外周神經損傷對GIRK通道四個亞單位的表達調控,分析和闡述GIRK通道在神經病理性疼痛發(fā)生和發(fā)展中所起到的作用。本研究利用免疫組織化學實驗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脊髓組織中的表達和分布情況。在正常大鼠DRG中~70%和10%的神經元分別表達GIRK1和GIRK2。利用降鈣素基因相關肽(CGRP)、植物凝集素B4(IB4)和神經絲蛋白-200(NF200)抗體分別標記DRG中的小型無髓鞘肽能神經元、小型無髓鞘非肽能神經元和中大型有髓鞘神經元,激光共聚焦共定位研究結果表明GIRK1和GIRK3廣泛地表達于這三種神經元類群,而GIRK2僅表達于CGRP和NF200陽性神經元類群。進一步使用四種鈣離子結合蛋白鈣結合蛋白D28k(CB)、鈣視網膜蛋白(CR)、小清蛋白(PV)和促泌素蛋白(Scgn)抗體標記不同的DRG神經元類群,對GIRK通道亞單位的表達特性進行進行地分析,結果表明GIRK1與四種鈣離子結合蛋白均存在不同程度上的共定位關系,而GIRK2只在CB陽性神經元中表達。為分析GIRK1~4在脊髓背角中的表達特性,使用不同的標記物分別標記DRG神經元軸突在脊髓背角投射末端分布的薄層(lamina),其中,GIRK1和GIRK2主要表達于lamina II,GIRK3和GIRK4主要表達于lamina I和外層lamina II。此外,GIRK1~4與VGLUT1或VGLUT2的共定位研究結果表明GIRK1~4在脊髓背角存在突觸前膜定位。利用神經元標記物Neu N標記脊髓背角神經元,結果表明GIRK1和GIRK2廣泛地表達于不同lamina區(qū)域的局部神經元,但是GIRK3和GIRK4不表達于局部神經元。為研究外周神經損傷對GIRK通道亞單位表達調控,我們通過大鼠坐骨神經離斷(axotomy)手術制備了外周神經損傷引起的神經病理性疼痛模型。通過免疫組織化學實驗、原位雜交實驗、實時熒光定量PCR實驗、western blot實驗和定量分析等方法對GIRK1~4在mRNA和蛋白質水平上的表達情況進行分析。結果表明損傷側DRG和脊髓背角中GIRK1、GIRK2和GIRK4的表達均顯著下調,而GIRK3的表達顯著上調。結合此前的研究結果進行分析,GIRK3表達的上調能夠降低神經元細胞膜的GIRK1/2和GIRK2/2通道。為研究GIRK通道亞單位在DRG神經元中的軸突轉運,分別利用大鼠坐骨神經結扎(ligation)模型和脊髓神經背根切除(rhizotomy)模型研究在DRG神經元中合成的GIRK1~4向外周端和中樞端運輸情況。實驗結果表明GIRK1~4均存在向外周和中樞運輸的順行轉運,此外,GIRK1~3存在外周軸突末端向胞體端運輸的逆行轉運。利用免疫組織化學實驗,進一步檢測了GIRK1~4在正常大鼠坐骨神經和后爪無毛皮膚中的表達和分布,結果表明GIRK1~4廣泛地表達于包括感覺纖維在內的多種類群的神經纖維,且GIRK1~4均表達于后爪無毛皮膚真皮層中的神經末端。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和脊髓中表達的神經化學特性,并通過外周神經損傷模型和多種分子生物學實驗方法證明了GIRK通道在神經病理性疼痛情況下的表達下調。GIRK通道表達的下調會引起感覺神經元的過度興奮,可能是神經病理性疼痛產生和長時程維持的重要原因。
[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.

【學位授予單位】：哈爾濱工業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R741

【相似文獻】

相關期刊論文 前10條

1 陳曦;陳定章;鄭敏娟;宋宏萍;周曉東;;實時超聲彈性成像評估外周神經損傷[J];中國醫(yī)學影像技術;2013年12期

2 李峰,穆廣態(tài),俞瑋,梁定順,康志學;外周神經損傷的顯微外科修復[J];中華顯微外科雜志;2004年01期

3 申東彥,任有成,袁正江,崔永光;顯微外科技術修復外周神經損傷126例[J];河南科技大學學報(醫(yī)學版);2004年04期

4 楊慶民,費紹波,叢海波,李金晟,畢衛(wèi)偉;外周神經損傷的臨床治療[J];中華顯微外科雜志;2005年03期

5 徐志強,戴國鵬;外周神經損傷手術探查的療效觀察[J];右江醫(yī)學;2005年01期

6 金淑萍;;手術治療外周神經損傷52例臨床分析[J];亞太傳統(tǒng)醫(yī)藥;2008年06期

7 何東升;;外周神經損傷的臨床特點與手術治療分析[J];中國現代醫(yī)生;2009年10期

8 江瀾;;外周神經損傷康復治療及肌電圖分析[J];中國康復;2010年04期

9 王愉思,許自力,王家讓;外周神經損傷顯微手術治療68例臨床分析[J];中華顯微外科雜志;2002年04期

10 馮經旺;湯克滬;馮運華;尹知訓;肖志林;沈景輝;譚志偉;林智峰;;延遲一期顯微手術修復外周神經損傷[J];中華顯微外科雜志;2006年02期

相關會議論文 前10條

1 陳曦;陳定章;鄭敏娟;宋宏萍;周曉東;;實時超聲彈性成像評估外周神經損傷的探討[A];中華醫(yī)學會第十三次全國超聲醫(yī)學學術會議論文匯編[C];2013年

2 張煥杰;;犬四肢外周神經損傷39例治療體會[A];中國畜牧獸醫(yī)學會2010年學術年會——第二屆中國獸醫(yī)臨床大會論文集(上冊)[C];2010年

3 呂藝;劉淑紅;葛學銘;范明;;不同類型外周神經損傷后神經元基因表達的比較[A];中國生理學會第21屆全國代表大會暨學術會議論文摘要匯編[C];2002年

4 項紅兵;肖建斌;戴雙華;招偉賢;;圍手術期外周神經損傷的評估和中醫(yī)藥治療[A];中國中西醫(yī)結合學會圍手術期專業(yè)委員會成立大會暨第二屆全國中西醫(yī)結合圍手術期醫(yī)學專題研討會論文集[C];2007年

5 張旭;;外周神經損傷后背根節(jié)神經元新表型及慢性痛分子機制研究[A];面向21世紀的科技進步與社會經濟發(fā)展（下冊）[C];1999年

6 陳定章;;外周神經損傷疑難病例分析[A];中華醫(yī)學會第十三次全國超聲醫(yī)學學術會議論文匯編[C];2013年

7 史南寧;李子淵;葉春嫦;黃雪麗;林志雄;楊秀容;;3例正己烷致嚴重外周神經損傷的報道[A];2011廣東省預防醫(yī)學會學術年會資料匯編[C];2011年

8 肖華勝;韓澤廣;張方雄;黃秋花;陸瑩謹;鮑嵐;陳竺;張旭;;正常和外周神經損傷后大鼠背根節(jié)基因表達譜的建立及差異基因的克隆[A];中國生物工程學會第三次全國會員代表大會暨學術討論會論文摘要集[C];2001年

9 胡杰;黃分;肖華勝;張旭;鮑嵐;;炎癥和外周神經損傷在背根節(jié)神經元中P2Y_1介導的反應增強[A];中國生理學會第六屆全國青年生理學工作者學術會議論文摘要[C];2003年

10 毛宇湘;牛黎明;陳澤;趙學民;馬擇錄;劉洪波;田克友;王學平;;中醫(yī)藥內服外洗治療AIDS抗病毒藥所致外周神經損傷的臨床研究(論文摘要)[A];中華中醫(yī)藥學會防治艾滋病分會第六屆學術年會論文匯編[C];2008年

相關博士學位論文 前5條

1 呂闖;外周神經損傷對DRG和脊髓神經細胞GIRK家族蛋白表達的影響[D];哈爾濱工業(yè)大學;2016年

2 周海洋;外周神經損傷對中樞核團的影響及骨髓間充質干細胞治療外周神經損傷后中樞逆行性病變的可行性研究[D];中南大學;2011年

3 劉素芳;缺氧及基質細胞衍生因子對神經干細胞移植治療外周神經損傷的影響[D];鄭州大學;2013年

4 呂藝;中樞與外周神經損傷后神經元胞體反應性的研究[D];中國人民解放軍軍事醫(yī)學科學院;2002年

5 肖華勝;外周神經損傷后大鼠背根節(jié)中基因的克隆和表達[D];第四軍醫(yī)大學;1999年

相關碩士學位論文 前2條

1 逄海東;局部微環(huán)境（免疫學）改變對外周神經損傷修復影響的實驗研究[D];青島大學;2011年

2 沈朝;含LacZ基因重組腺病毒的制備及其在大鼠脊髓中的表達[D];河北醫(yī)科大學;2011年

，

本文編號：1508210