【摘要】：腎上腺髓質(zhì)素(adrenomedullin, AM)屬于降鈣素基因相關(guān)肽(calcitonin gene-related peptide, CGRP)家族,廣泛存在于脊髓背角淺層與背根神經(jīng)節(jié)(DRG)的肽能和非肽能神經(jīng)元。研究指出,AM是一種痛介導(dǎo)質(zhì),但其參與慢性神經(jīng)痛的機制尚未清晰。為探索AM在神經(jīng)病理性痛發(fā)生和維持過程的作用機制,以及與CGRP、白細胞介素-1β(interleukin 1β, IL-1β)、NO之問的關(guān)系,本研究建立在骨癌痛、L5脊神經(jīng)結(jié)扎SNL基礎(chǔ)上,采用影像學(xué)、行為學(xué)、免疫組織化學(xué)和實時熒光定量PCR (quantitative real-time polymerase chain reaction, qRT-PCR)等實驗技術(shù)探討：(1)骨癌引起大鼠組織學(xué)、行為學(xué)的變化；(2)骨癌痛誘發(fā)大鼠脊髓背角和DRG中AM、CGRP和nNOS的表達情況；(3)AM特異性受體拮抗劑AM22-52對骨癌誘發(fā)大鼠痛覺過敏和AM、CGRP、NOS表達的影響；(4)SNL大鼠機械痛閾值的變化以及AM、CGRP、IL-1β的表達情況；(5)鞘內(nèi)注射AM22-52對SNL大鼠行為學(xué)的影響；(6)鞘內(nèi)注射AM22-52 對 SNL誘發(fā)大鼠脊髓和DRG中AM、CGRP、IL-1β表達的影響。實驗結(jié)果如下：(1)骨癌大鼠第7天出現(xiàn)體重下降、明顯的骨破壞現(xiàn)象,并產(chǎn)生機械痛覺過敏和熱痛覺過敏。(2)骨癌痛引起AM及其受體、CGRP和nNOS在脊髓背角和DRG中表達增加,染色顯示AM主要分布于DRG中、小型神經(jīng)元。(3)鞘內(nèi)注射AM22-52,翻轉(zhuǎn)了骨癌痛誘發(fā)的痛覺過敏及脊髓背角和DRG內(nèi)AM、CGRP、NOS的表達增加。(4)大鼠脊神經(jīng)結(jié)扎處理后第10天機械閾值下降50%左右,產(chǎn)生痛覺過敏。DRG和脊髓背角中AM、CGRP、IL-1β的表達明顯增加。(5)鞘內(nèi)注射AM22-52可緩解SNL引發(fā)的痛覺過敏。(6)鞘內(nèi)注射AM22-52可明顯緩解脊髓和L4中CGRP表達增加,而AM和IL-1p表達量反而升高。以上研究結(jié)果表明,骨痛和神經(jīng)損傷能誘導(dǎo)AM合成增加,阻斷AM受體可明顯緩解疼痛；神經(jīng)病理性痛誘導(dǎo)的細胞信號級聯(lián)反應(yīng)中,AM可能是使促癇介質(zhì)CGRP和促炎性細胞因子IL-1β、nNOS等表達增加的上游因子。與此同時,除AM、CGRP通路以外,可能存在免疫機制或其他潛在的信號通路誘導(dǎo)促炎因子IL-1β的表達,參與疼痛的發(fā)生。
[Abstract]:Adrenomedullin (AM) belongs to the calcitonin gene-related peptide (CGRP) family and widely exists in peptidergic and non-peptidergic neurons in the superficial layer of spinal dorsal horn and dorsal root ganglion (DRG). This study was based on bone cancer pain, L5 spinal nerve ligation SNL, using imaging, behavioral, immunohistochemical and quantitative real-time polymerase chain reaction (quantitative real-time polymerase chain reaction). Experimental techniques such as ain reaction and qRT-PCR were used to investigate: (1) histological and behavioral changes induced by bone cancer in rats; (2) expression of AM, CGRP and nNOS in spinal dorsal horn and DRG induced by bone cancer pain in rats; (3) effect of AM22-52 on hyperalgesia and expression of AM, CGRP and NOS induced by bone cancer in rats; (4) mechanical pain in SNL rats The changes of the threshold and the expression of AM, CGRP and IL-1 beta were observed; (5) the effect of intrathecal injection of AM22-52 on the behavior of SNL rats; (6) the effect of intrathecal injection of AM22-52 on the expression of AM, CGRP and IL-1 beta in spinal cord and DRG of rats induced by SNL. (3) Intrathecal injection of AM22-52 reversed the hyperalgesia induced by bone cancer pain and increased the expression of AM, CGRP and NOS in spinal dorsal horn and DRG in rats. (4) The expression of AM, CGRP and NOS in spinal nerve node was increased in rats. On the 10th day after ligation, the mechanical threshold decreased by about 50%, resulting in hyperalgesia. The expression of AM, CGRP and IL-1 beta in DRG and dorsal horn of spinal cord increased significantly. (5) Intrathecal injection of AM22-52 could alleviate the hyperalgesia induced by SNL. (6) Intrathecal injection of AM22-52 could significantly alleviate the increase of CGRP expression in spinal cord and L4, while the expression of AM and IL-1p increased. The results showed that bone pain and nerve injury could induce the increase of AM synthesis and blockade of AM receptor could alleviate pain. AM may be an upstream factor in the signal cascade reaction induced by neuropathic pain, which may increase the expression of CGRP, IL-1beta, nNOS and other inflammatory cytokines. The mechanism or other potential signaling pathways induce the expression of proinflammatory cytokines IL-1 beta and participate in the occurrence of pain.
【學(xué)位授予單位】：福建師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R402

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