【作者】 陈梦玲；

【导师】 赵志奇；

【作者基本信息】 复旦大学 ， 神经生物学， 2010， 博士

【摘要】 吗啡耐受是临床控制疼痛的一大障碍,其机制的研究已有很长历史。近年来的研究表明,脊髓小胶质细胞在吗啡耐受过程中具有重要作用。但是关于小胶质细胞参与吗啡耐受的作用机制尚未阐明。P2X7受体(P2X7R),作为ATP受体P2X家族的一种亚型,属于ATP门控的离子通道受体。它在身体各个组织中有比较广泛的分布,主要表达在免疫系统。在中枢神经系统中,小胶质细胞作为免疫源性细胞,也表达大量的P2X7R。P2X7R的功能不仅涉及多种促炎因子(proinflammatory cytokines)的合成和释放,还对谷氨酸(glutamate)的释放和再摄取具有调控作用。最近有研究显示,神经病理性疼痛引起脊髓P2X7R表达明显上调,而干扰P2X7R的功能可以减轻疼痛。鉴于吗啡镇痛耐受与神经病理性疼痛形成机制上的相似性,提示脊髓P2X7R可能参与吗啡镇痛耐受的形成。本实验采用行为学、细胞培养、Western blot、免疫荧光以及分子生物学技术,在吗啡镇痛耐受的大鼠模型上,对脊髓P2X7R的作用进行了研究,并对其机制进行了探讨。取得的主要结果如下：1.脊髓小胶质细胞中P2X7R对慢性吗啡镇痛耐受形成和维持的影响对胚胎大鼠(孕17d)脊髓原代混合培养细胞以及正常或吗啡耐受成年大鼠脊髓切片进行免疫荧光染色的结果显示,P2X7R仅分布在OX42阳性的小胶质细胞,而不位于星形胶质细胞或神经元。大鼠脊髓P2X7R蛋白水平随着吗啡耐受的形成而逐渐增加,在给药后第4d出现统计学差异。鞘内预先给予P2X7R的拮抗剂(oxATP或BBG)或针对P2X7R的小干扰RNA片段(P2X7siRNA)均能对吗啡镇痛耐受的形成产生抑制作用。在已经建立了吗啡镇痛耐受的实验大鼠上,每天皮下注射吗啡前30 min鞘内预先给予BBG,连续3天,对已经减弱的吗啡镇痛效能没有改善。提示,脊髓P2X7R参与慢性吗啡镇痛耐受的形成,但不参与耐受的维持。2.脊髓P2X7R在吗啡耐受过程中增强神经元与胶质细胞的活化脊髓小胶质细胞标志物Iba1、神经元激酶PKCγ与星形胶质细胞标志物GFAP的蛋白随着吗啡镇痛耐受的形成均出现表达增加,分别在吗啡注射的第2d、第4d及第6d与对照组相比出现统计学差异。鞘内预先给予BBG抑制P2X7R的功能或者给予P2X7 siRNA预先下调P2X7R的表达,均可显著抑制由长期吗啡注射引起的脊髓小胶质细胞中Iba1或p-p38的蛋白表达增加,同时抑制由慢性吗啡引起的GFAP及PKCγ的蛋白水平增高。这些结果表明,脊髓P2X7R通过影响神经元与胶质细胞的活化介导吗啡耐受。3.脊髓促炎因子IL-18在P2X7R介导的吗啡耐受中的作用大鼠脊髓中促炎症因子IL-18及其受体IL-18R的蛋白水平随着吗啡镇痛耐受的形成均逐渐增加,分别在吗啡注射的第4d和第6d与对照组相比出现统计学差异。形态学研究显示,IL-18主要位于脊髓小胶质细胞；而IL-18R主要位于星形胶质细胞,少量分布于神经元。鞘内预先给予拮抗IL-18功能的IL-18BP或IL-18Ab均能显著抑制吗啡耐受的形成。鞘内预先给予BBG或者P2X7 siRNA均能显著抑制慢性吗啡引起的脊髓IL-18、IL-1β以及pNFκB的表达增加。而鞘内预先给予IL-18BP,也可以显著抑制由长期吗啡注射引起GFAP及PKCγ的蛋白水平增高,但对小胶质细胞标志物Iba1或星形胶质细胞中pNFκB的表达变化没有显著影响。从而提示,脊髓小胶质细胞的促炎因子IL-18可能作为P2X7R下游靶点之一,通过介导星形胶质和神经元的激活而参与吗啡耐受过程。此外,NFκB参与了P2X7R介导的吗啡耐受过程,但不作为IL-18影响星形胶质细胞活化的主要通路。综上所述,本研究首次揭示脊髓小胶质细胞特异性表达的P2X7R在吗啡镇痛耐受发展过程中发生了显著地改变,并且对吗啡镇痛耐受的产生而非维持有着重要的作用。此外,促炎因子IL-18作为其可能的下游机制之一,介导小胶质细胞-星形胶质细胞-神经元的级联反应,影响它们的活化,从而参与吗啡耐受的形成。这些结果拓展了胶质细胞参与吗啡镇痛耐受的可能机制,并且为临床上改善吗啡的长期镇痛效能提供了一个潜在靶点。更多还原

【Abstract】 Opioid substances, like morphine, are the most effective analgesics used to treat moderate to severe pain. However, their use is limited by the development of tolerance, which is manifested clinically by the need for increasing opioid dosages over time to maintain the same level of pain relief. Recently, the role of microglia in the central nervous system in the development of analgesic tolerance has been studied. Although considerable progress has been made, the mechanism underlying microglial contribution to morphine analgesic tolerance is not fully explored.P2X7 receptor (P2X7R) is a subtype of the ATP-gated non-selective ion channel family P2X. It has a widespread tissue distribution and its expression is particularly strong in the immune system. In spinal cord, P2X7R is predominately present in microglia. P2X7R plays an important role in the production of proinflammatory cytokines and the regulation of glutamate transporter efficiency. Recently, mounting evidence indicates that P2X7R is involved in multiple neuropathic models. Given that morphine analgesic tolerance and neuropathic pain are supposed to share some common mechanisms, it is reasonable to hypothesize that spinal P2X7R contributes to morphine analgesic tolerance.Here, we tested our hypothesis in rats using experimental techniques including behavioral tests, RNA interference, Western blot analysis, immunofluorescence and cell culture. The results were as follows:1. Spinal microglia-expressed P2X7R contributed to the development of chronic morphine tolerance but not the maintenanceP2X7Rs were exclusively localized in OX42-labeled microglia in spinal dorsal horn revealed by immunofluorescence, which was performed on lumbar spinal cord sections from normal and morphine tolerated adult rats. The cellular localization of P2X7R was confirmed in the mixed-glia culture from 17-day-old embryonic (E17) SD rats. The expression of spinal P2X7R was upregulated gradually during the development of morphine analgesic tolerance; and the significant increase occurred since D4 (after 3 days’morphine injection), at which tolerance was just generated. Pretreatment with P2X7R antagonists [oxidized ATP (oxATP) or brilliant blue G (BBG)] or P2X7R-targetting siRNA intrathecally inhibited the development of chronic morphine tolerance significantly. However, intrathecal administration of BBG failed to reverse the established tolerance to morphine analgesia.2. Spinal P2X7R mediated the activation of glia and neurons in the development of morphine analgesic toleranceThe expressions of microglial marker Ibal, the neuronal kinase PKCy and astroglial marker GFAP in rat lumbar spinal cord were upregulated upon the development of chronic morphine tolerance; and the significant increase began at D2, D4 and D6 respectively. Pretreatment with P2X7R antagonist BBG or P2X7R-targetting siRNA intrathecally not only inhibited the upregulation of Ibal and p-p38 expression in microglia, but also suppressed the upregulation of GFAP expression in astroglia and PKCy expression in neurons of the spinal cord treated by chronic morphine.3. Spinal proinflammatory cytokine interleukin 18 (IL-18) as one downstream of P2X7R was involved in chronic tolerance to morphine analgesiaThe expressions of proinflammatory cytokine IL-18 and its corresponding receptor IL-18 receptor (IL-18R) in rat lumbar spinal cord were upregulated upon the development of chronic morphine tolerance; and the significant increase began at D4 and D6 respectively. IL-18 was mainly localized in OX42-positive microglia of rat spinal cord, whereas IL-18R majorly distributed in GFAP-labeled astroglia. Pretreatment with IL-18 antagonists IL-18BP or IL-18Ab intrathecally inhibited the development of morphine tolerance significantly. Pre-administration of P2X7R antagonist BBG or P2X7R-targetting siRNA intrathecally suppressed the upregulation of spinal IL-18, IL-1βand pNFκB induced by repeated morphine injection remarkably. Pretreatment with IL-18BP intrathecally suppressed the upregulation of spinal GFAP and PKCy significantly, but not affected the upregulation of either Ibal or pNFκB induced by chronic morphine.Taken together, we demonstrated that the expression of spinal microglia-expressed P2X7R was increased after chronic exposure to morphine, which facilitated the development but not the maintenance of morphine analgesic tolerance. Moreover, as one of P2X7R downstream targets, pro inflammatory cytokine IL-18 was involved in the generation of morphine tolerance, possibly by mediating the activation of microglia-astroglia-neurons cascade. Although NFκB was involved in the downstream of P2X7R, it might not be the predominant pathway through which IL-18 mediated the activation of astroglia. These findings highlight the possibility of a new clinical strategy to improve morphine tolerance.更多还原