【摘要】：腎上腺髓質(zhì)素(adrenomedullin, AM)是降鈣素基因相關(guān)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(jué)神經(jīng)元都有表達(dá),是一種疼痛相關(guān)肽。最近的研究發(fā)現(xiàn),慢性應(yīng)用嗎啡,通過(guò)μ-阿片受體和蛋白激酶C信號(hào)通路導(dǎo)致AM表達(dá)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(jìn)嗎啡耐受的細(xì)胞學(xué)機(jī)制尚不清楚。為揭示AM參與嗎啡耐受的細(xì)胞學(xué)機(jī)制。本研究通過(guò)在體和離體實(shí)驗(yàn),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(yīng)用嗎啡誘導(dǎo)的胞內(nèi)痛介導(dǎo)質(zhì)興奮性氨基酸和促炎性細(xì)胞因子的變化。并采用行為學(xué)、實(shí)時(shí)熒光定量PCR、蛋白印跡、免疫熒光組化等技術(shù)研究AM調(diào)節(jié)促炎性細(xì)胞因子的細(xì)胞學(xué)機(jī)制,揭示AM參與嗎啡耐受的機(jī)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(mén)冬氨酸)釋放增加,還能抑制脊髓背角中促炎性細(xì)胞因子IL-1β、IL-6mRNA表達(dá)增加。(2) AM siRNA (50nM)干擾離體培養(yǎng)的DRG外植體,能抑制慢性應(yīng)用嗎啡(3.3μM)誘導(dǎo)的DRG外植體中促炎性細(xì)胞因子IL-1β、IL-6mRNA表達(dá)增加。(3)單獨(dú)連續(xù)7天鞘內(nèi)注射AM受體激動(dòng)劑AM1-50(10μg),能引起大鼠脊髓背角中IL-1β、IL-6和TNF-α mRNA表達(dá)增加,卻引起DRG中IL-1β、IL-6和TNFα mRNA表達(dá)下降。(4)單獨(dú)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(xì)胞和小膠質(zhì)細(xì)胞特異性標(biāo)志物GFAP和OX-42表達(dá)增加；而鞘內(nèi)注射AM22-52,能抑制慢性應(yīng)用嗎啡誘導(dǎo)的大鼠脊髓背角GFAP和OX-42表達(dá)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(xì)胞標(biāo)志物OX-42共表達(dá)。(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(dá)增加；鞘內(nèi)聯(lián)合注射小膠質(zhì)細(xì)胞選擇性抑制劑米諾環(huán)素可抑制慢性應(yīng)用AM1-s0誘發(fā)的痛覺(jué)過(guò)敏,抑制p38磷酸化水平增加,抑制促炎性細(xì)胞因子IL-1β、IL-6、TNF-α mRNA表達(dá)增加。 以上結(jié)果表明：AM受體通過(guò)調(diào)節(jié)興奮性氨基酸(谷氨酸和天門(mén)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(guò)直接方式引起星型膠質(zhì)細(xì)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(jī)制之一是通過(guò)小膠質(zhì)細(xì)胞p38MAPK信號(hào)通路介導(dǎo)。
[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(xué)位授予單位】：福建師范大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R96

【共引文獻(xiàn)】

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