本文選題：亨廷頓蛋白相關(guān)蛋白1 + 辣椒素��； 參考：《華中科技大學(xué)》2006年碩士論文

【摘要】： 亨廷頓蛋白相關(guān)蛋白1(huntingtin-associated protein 1,HAP1)是一種功能未明的蛋白質(zhì),因其與亨廷頓病(Huntington’s disease, HD)基因產(chǎn)物亨廷頓蛋白(huntingtin)具有相互作用而被確定為第一個(gè)亨廷頓蛋白相關(guān)蛋白。HAP1至少具有2種同工異構(gòu)型(isoforms),即HAP1A和HAP1B。HAP1廣泛分布于神經(jīng)系統(tǒng)內(nèi),其中HAP1A主要定位于神經(jīng)元胞體和軸突終末內(nèi),而HAP1B主要定位于神經(jīng)元胞體和樹突內(nèi)。本實(shí)驗(yàn)室以前的研究發(fā)現(xiàn),HAP1在大鼠脊髓背角淺層有高水平表達(dá),而在脊髓腹角的表達(dá)水平極低,外周疼痛刺激可使脊髓背角淺層內(nèi)HAP1及其mRNA表達(dá)水平升高,由此提示脊髓背角內(nèi)的HAP1可能參與感覺信息特別是傷害性信息(痛覺)的初級(jí)傳入和/或調(diào)控。為了證明這一假設(shè),本研究應(yīng)用免疫電鏡技術(shù)觀察初級(jí)傳入C纖維(簡(jiǎn)稱C纖維)與脊髓背角內(nèi)HAP1神經(jīng)元的突觸聯(lián)系,并應(yīng)用免疫印跡和RT-PCR技術(shù)檢測(cè)C纖維是否參與外周疼痛刺激促進(jìn)脊髓背角內(nèi)HAP1及其mRNA表達(dá)的過(guò)程。 對(duì)正常大鼠腰段脊髓進(jìn)行HAP1B的ABC法免疫組織化學(xué)染色,光鏡下觀察顯示,HAP1免疫反應(yīng)產(chǎn)物主要分布在脊髓灰質(zhì)背角淺層神經(jīng)元胞體和神經(jīng)氈內(nèi),灰質(zhì)其他各層無(wú)或極少HAP1B表達(dá),白質(zhì)內(nèi)未見明顯HAP1B免疫反應(yīng)產(chǎn)物。經(jīng)成年大鼠寰枕骨下穿刺蛛網(wǎng)膜下腔插管注射辣椒素到腰段脊髓特異性損毀C纖維,7天后取腰段脊髓進(jìn)行HAP1B的ABC法免疫電鏡標(biāo)記。電鏡下觀察發(fā)現(xiàn),在蛛網(wǎng)膜下腔注射辣椒素大鼠脊髓背角淺層內(nèi),大量變性的C纖維軸突終末表現(xiàn)為囊泡結(jié)構(gòu)模糊不清,軸漿基質(zhì)內(nèi)電子密度均勻增高;HAP1B免疫反應(yīng)產(chǎn)物主要分布在神經(jīng)元胞體和樹突內(nèi),并可見變性軸突終末與1至多個(gè)HAP1B陽(yáng)性樹突形成非對(duì)稱性軸樹突觸或突觸球。 免疫印跡和RT-PCR檢測(cè)顯示,在新生期(出生后2天)皮下注射辣椒素的成年大鼠和成年期蛛網(wǎng)膜下腔注射辣椒素7天的大鼠,單側(cè)疼痛刺激(足底注射10%福爾馬林)24h后,注射側(cè)脊髓背角內(nèi)HAP1B和其mRNA的表達(dá)水平與對(duì)照側(cè)無(wú)明顯差異,而在新生期皮下注射溶媒的成年大鼠,疼痛刺激側(cè)脊髓背角內(nèi)HAP1B和其mRNA的表達(dá)水平均較對(duì)照側(cè)明顯增高。 辣椒素是一種對(duì)C纖維具有選擇性毒性神經(jīng)毒素,在新生期皮下注射辣椒素或在成年期蛛網(wǎng)膜下腔注射辣椒素均可使C纖維大量潰變或變性,是一種公認(rèn)的破環(huán)C纖維的工具藥。本實(shí)驗(yàn)中,在蛛網(wǎng)膜下腔注射辣椒素的大鼠脊髓背角淺層內(nèi)C纖維變性終末與HAP1B陽(yáng)性的樹突間非對(duì)稱性的軸樹突觸或突觸球的存在證明,脊髓背角中的HAP1B神經(jīng)元能被C纖維傳入的傷害性信號(hào)激活;疼痛刺激能促進(jìn)脊髓背角中HAP1B和其mRNA的表達(dá),而在辣椒素毀損C纖維后,疼痛刺激促進(jìn)脊髓背角中HAP1B和其mRNA表達(dá)的作用不再存在或明顯減弱,這現(xiàn)象則表明,C纖維參與外周疼痛刺激促進(jìn)脊髓背角內(nèi)HAP1及其mRNA表達(dá)的過(guò)程。脊髓背角中的HAP1在外周傷害性刺激通過(guò)C纖維的興奮作用而表達(dá)增加后可能參與對(duì)傷害性信息傳入的調(diào)節(jié)過(guò)程。
[Abstract]:Huntington protein related protein 1 (huntingtin-associated protein 1, HAP1) is an unfunctional protein that has been identified as the first Huntington protein related protein.HAP1 with at least 2 types of identical isomer because of its interaction with the Huntington disease (Huntington 's disease, HD) gene product Huntington protein (huntingtin). RMS), that is, HAP1A and HAP1B.HAP1 are widely distributed in the nervous system, in which HAP1A is located mainly in the neuronal cell body and the end of the axon, while HAP1B is located mainly in the neurons and dendrites. Previous studies in this laboratory found that HAP1 was highly expressed in the shallow layer of the dorsal horn of the spinal cord of the rat, and the expression level in the ventral horn of the spinal cord was very low, and the peripheral part of the spinal cord was very low. Pain stimulation can increase the level of HAP1 and mRNA in the superficial dorsal horn of the spinal cord, suggesting that HAP1 in the dorsal horn of the spinal cord may be involved in primary afferent and / or regulation of sensory information, especially nociceptive information (pain sensation). In order to prove this hypothesis, this study applied immuno electron microscopy to observe the primary afferent C fiber (C fiber) and the spinal cord. The synapse of HAP1 neurons in the dorsal horn and the immunoblotting and RT-PCR techniques were used to detect whether C fibers were involved in peripheral pain stimulation to promote the expression of HAP1 and mRNA in the dorsal horn of the spinal cord.
ABC immunohistochemical staining of HAP1B in the spinal cord of normal rats was carried out. The results of light microscopy showed that the products of HAP1 immunoreaction were mainly distributed in the shallow layer neurons of the spinal gray matter and the nerve felt in the shallow layer of the gray matter of the spinal cord. There was no or little HAP1B expression in the other layers of the gray matter, and there was no obvious HAP1B immunoreactivity in the white matter. Under the subarachnoid subarachnoid catheterization, capsaicin was injected into the lumbar spinal cord with specific damage to the C fiber, and the lumbar spinal cord was taken for HAP1B by ABC immunoelectron microscopy 7 days later. Under the electron microscope, it was found that in the subarachnoid injection of capsaicin in the superficial layer of the spinal dorsal horn of the capsaicin, a large number of denatured C fiber axons were characterized by the ambiguous structure of the vesicles. The electron density in the pulp matrix increased evenly, and the HAP1B immunoreaction products were mainly distributed in the neurons and dendrites, and the denatured axon terminals formed asymmetric axonal synapses or synapses with 1 to a number of HAP1B positive dendrites.
Western blot and RT-PCR showed that the expression level of HAP1B and mRNA in the dorsal horn of the spinal cord was not significantly different from the control side, but there was no significant difference in the expression level of the HAP1B and its mRNA in the dorsal horn of the spinal cord after the subcutaneous injection of capsaicin and the subarachnoid injection of capsaicin for 7 days in the adult rat and the subarachnoid injection of the subarachnoid cavity. In adult rats, the expression levels of HAP1B and mRNA in the spinal cord dorsal horn were significantly higher than those in the control side.
Capsaicin is a selective toxic neurotoxin for C fibers. Capsaicin injection in the newborn period, or capsaicin injection in the subarachnoid space of adulthood, can make C fiber a large amount of degeneration or denaturation. It is a recognized tool for breaking the C fiber. In this experiment, C in the shallow layer of the spinal dorsal horn of the subarachnoid injection of capsaicin in rats The presence of asymmetrical axisymmetric synapses or synapses between fiber degeneration and HAP1B positive dendrites proves that HAP1B neurons in the dorsal horn of the spinal cord can be activated by the nociceptive signals transmitted by C fibers; pain stimulation can promote the expression of HAP1B and mRNA in the dorsal horn of the spinal cord, and the pain stimulates the dorsal horn of the spinal cord after the paprika destroys the C fiber. The expression of HAP1B and its mRNA no longer exists or significantly diminished, which indicates that the involvement of C fibers in peripheral pain stimulates the expression of HAP1 and its mRNA in the dorsal horn of the spinal cord. HAP1 in the dorsal horn of the spinal cord may be involved in the regulation of nociceptive information after the increase of the expression of the peripheral nociceptive stimulation through the excitation of the C fiber. Process.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R329

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本文編號(hào)：2015958