【摘要】：腎上腺髓質(zhì)素(adrenomedullin, AM)是降鈣素基因相關肽(calcitonin gene related peptide, CGRP)家族的一員,與CGRP具有相似的功能。AM在脊髓背角淺層和背根神經(jīng)節(jié)感覺神經(jīng)元都有表達,是一種疼痛相關肽。最近的研究發(fā)現(xiàn),慢性應用嗎啡,通過μ-阿片受體和蛋白激酶C信號通路導致AM表達上調(diào),引起嗎啡耐受,鞘內(nèi)注射AM受體拮抗劑AM22-52可翻轉(zhuǎn)嗎啡耐受。這一結(jié)果提示,AM參與了嗎啡耐受的形成。但是其促進嗎啡耐受的細胞學機制尚不清楚。為揭示AM參與嗎啡耐受的細胞學機制。本研究通過在體和離體實驗,分別采用AM受體拮抗劑和AMsiRNA抑制AM受體,觀察阻斷AM受體功能后,慢性應用嗎啡誘導的胞內(nèi)痛介導質(zhì)興奮性氨基酸和促炎性細胞因子的變化。并采用行為學、實時熒光定量PCR、蛋白印跡、免疫熒光組化等技術研究AM調(diào)節(jié)促炎性細胞因子的細胞學機制,揭示AM參與嗎啡耐受的機制。 結(jié)果顯示：(1)鞘內(nèi)給予AM受體拮抗劑AM22-52(10nmol),能抑制嗎啡耐受大鼠脊髓背角和血漿中興奮性氨基酸(谷氨酸和天門冬氨酸)釋放增加,還能抑制脊髓背角中促炎性細胞因子IL-1β、IL-6mRNA表達增加。(2) AM siRNA (50nM)干擾離體培養(yǎng)的DRG外植體,能抑制慢性應用嗎啡(3.3μM)誘導的DRG外植體中促炎性細胞因子IL-1β、IL-6mRNA表達增加。(3)單獨連續(xù)7天鞘內(nèi)注射AM受體激動劑AM1-50(10μg),能引起大鼠脊髓背角中IL-1β、IL-6和TNF-α mRNA表達增加,卻引起DRG中IL-1β、IL-6和TNFα mRNA表達下降。(4)單獨連續(xù)7天鞘內(nèi)注射AM1-50,能引起脊髓背角星型膠質(zhì)細胞和小膠質(zhì)細胞特異性標志物GFAP和OX-42表達增加；而鞘內(nèi)注射AM22-52,能抑制慢性應用嗎啡誘導的大鼠脊髓背角GFAP和OX-42表達增加。(5)免疫熒光雙重染色顯示在大鼠脊髓背角中AM受體的結(jié)合部件CRLR和RAMP2與小膠質(zhì)細胞標志物OX-42共表達。(6)連續(xù)7天鞘內(nèi)注射AM1-50,可引起脊髓背角磷酸化p38蛋白表達增加；鞘內(nèi)聯(lián)合注射小膠質(zhì)細胞選擇性抑制劑米諾環(huán)素可抑制慢性應用AM1-s0誘發(fā)的痛覺過敏,抑制p38磷酸化水平增加,抑制促炎性細胞因子IL-1β、IL-6、TNF-α mRNA表達增加。 以上結(jié)果表明：AM受體通過調(diào)節(jié)興奮性氨基酸(谷氨酸和天門冬氨酸)和促炎性因子IL-1β、IL-6和TNF-α合成參與嗎啡耐受。AM受體通過直接方式引起星型膠質(zhì)細胞和小膠質(zhì)激活,促使其增加IL-1β、IL-6和TNF-α合成,引起嗎啡耐受。其作用機制之一是通過小膠質(zhì)細胞p38MAPK信號通路介導。
[Abstract]:Adrenomedullin (adrenomedullin, AM) is a member of calcitonin gene-related peptide (calcitonin gene related peptide, CGRP) family. It has similar function with CGRP. AM is expressed in the sensory neurons of dorsal horn and dorsal root ganglion. It is a kind of pain related peptide. Recent studies have found that chronic morphine administration through 渭 -opioid receptor and protein kinase C signaling pathway leads to upregulation of AM expression, leading to morphine tolerance, and intrathecal injection of AM receptor antagonist AM22-52 can reverse morphine tolerance. These results suggest that AM is involved in the development of morphine tolerance. However, the cytological mechanism for promoting morphine tolerance is unclear. To elucidate the cytological mechanism of AM involved in morphine tolerance. In this study, in vivo and in vitro, AM receptor antagonists and AMsiRNA were used to inhibit AM receptor, respectively. After blocking the function of AM receptor, the changes of intracellular excitatory amino acids and pro-inflammatory cytokines induced by morphine were observed. The cytological mechanism of AM regulating pro-inflammatory cytokines was studied by means of behavioral, real-time fluorescent quantitative PCR, Western blotting and immunofluorescence histochemistry, and the mechanism of AM involved in morphine tolerance was revealed. The results showed that: (1) intrathecal administration of AM receptor antagonist AM22-52 (10nmol) inhibited the increased release of excitatory amino acids (glutamate and aspartate) in spinal dorsal horn and plasma of morphine tolerant rats. It also inhibited the expression of pro-inflammatory cytokine IL-1 尾 -mil 6 mRNA in spinal dorsal horn. (2) AM siRNA (50nM) interfered with DRG explants cultured in vitro. The expression of pro-inflammatory cytokine (IL-1 尾) and IL-6 mRNA in DRG explants induced by chronic morphine administration (3.3 渭 M) was inhibited. (3) Intrathecal injection of AM receptor agonist AM1-50 (10 渭 g),) alone for 7 days could increase the expression of IL-1 尾 IL-6 and TNF- 偽 mRNA in the dorsal horn of spinal cord of rats. However, the expression of IL-1 尾 -IL-6 and TNF 偽 mRNA decreased in DRG. (4) Intrathecal injection of AM1-50, alone for 7 days increased the expression of GFAP and OX-42, the specific markers of astrocyte and microglia in the dorsal horn of spinal cord. Intrathecal injection of AM22-52, inhibited the increase of GFAP and OX-42 expression in the spinal dorsal horn of rats induced by chronic morphine. (5) Immunofluorescence double staining showed that CRLR and RAMP2, the binding components of AM receptor, and microglia markers in the dorsal horn of spinal cord of rats. (6) Intrathecal injection of AM1-50, for 7 days increased the expression of phosphorylated p38 protein in the dorsal horn of spinal cord. Intrathecal injection of minocycline, a selective microglia inhibitor, could inhibit hyperalgesia induced by chronic AM1-s0, increase the level of p38 phosphorylation, and inhibit the expression of pro-inflammatory cytokine IL-1 尾, IL-6, TNF- 偽 mRNA. These results suggest that the receptor of 1: AM participates in the activation of astrocytes and microglia by regulating the synthesis of excitatory amino acids (glutamic acid and aspartic acid) and the pro-inflammatory factors IL-1 尾, IL-6 and TNF- 偽. It increased the synthesis of IL-1 尾 -IL-6 and TNF- 偽 and induced morphine tolerance. One of its mechanisms is mediated by microglial p38MAPK signaling pathway.
【學位授予單位】：福建師范大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R96

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