去甲肾上腺素α2受体对脊髓NMDA受体介导的痛觉突触传递的调节作用

发布时间：2018-06-05 07:24

本文选题：去甲肾上腺素α2受体 + NMDA受体　；参考：《兰州大学》2014年硕士论文

【摘要】：目的：去甲肾上腺素α2受体高度富集在脊髓背角。外周组织损伤发生时,鞘内注射去甲肾上腺素α2受体激动剂可以有效地缓解病理性疼痛。但其确切的发生机制,目前尚不十分清楚。NMDA(N-甲基-D-天冬氨酸)型谷氨酸受体功能亢进,被认为是诱发慢性病理性疼痛的核心机制之一。本课题的目标,在于探讨去甲肾上腺素α2受体对脊髓NMDA受体介导的痛觉突触传递的调节作用,并揭示其可能的分子机制。方法：本研究运用行为学测定、膜片钳电生理记录和免疫蛋白印迹等方法,探讨去甲肾上腺素α2受体激动剂对炎性疼痛小鼠脊髓背角NMDA受体的调节作用及其信号转导通路。结果：(1)小鼠后足底皮下注射完全弗氏佐剂(CFA)制备炎性疼痛模型,24h后制得脊髓切片,在背角II板层神经元上的全细胞记录结果显示,去甲肾上腺素α2受体激动剂Clonidine通过a2A亚型受体,显著地降低炎性疼痛小鼠NMDA受体介导的兴奋性突触后电流(NMDAR-EPSCs)的幅值；(2)记录、并计算双刺激比值(paired-pulse ratio; PPR;=2nd response/1st response)发现,细胞外灌流Clonidine前、后,PPR并不发生明显的变化,提示：Clonidine抑制炎性疼痛小鼠NMDAR-EPSCs的作用发生在突触后膜；(3)通过记录电极、向细胞内导入Gα抑制剂GDP-β-S,可明显阻断Clonidine对NMDAR-EPSCs的抑制作用；而直接抑制cAMP依赖蛋白激酶(PKA),也可以模拟Clonidine对NMDAR-EPSCs的抑制效应,提示：Clonidine可能通过“Gi蛋白/PKA信号通路”起效：(4)整体实验结果显示,鞘内注射Clonidine可特异性降低突触中包含GluN2B亚基的NMDA受体(即GluN2B受体)的含量：(5)离体电生理记录的结果进一步证明,GluN2B受体选择性抑制剂ifenprodil不仅能够降低炎性疼痛小鼠NMDA受体介导的突触传递,而且会完全饱和Clonidine的突触抑制效应,提示：去甲肾上腺素α2受体可能通过选择性下调GluN2B受体的突触功能而发挥镇痛作用。结论：去甲肾上腺素α2受体可能通Gi蛋白/PKA/GluN2B受体信号通路,抑制炎性疼痛小鼠脊髓背角中NMDA受体介导的痛觉突触传递,缓解慢性炎性疼痛症状。
[Abstract]:Objective: norepinephrine 伪 2 receptor is highly enriched in the dorsal horn of spinal cord. Intrathecal injection of norepinephrine 伪 2 receptor agonist can effectively relieve pathological pain during peripheral tissue injury. However, the exact mechanism of Glutamic acid receptor hyperfunction, which is considered to be one of the core mechanisms to induce chronic pain, is still unclear. The aim of this study is to investigate the regulatory effect of norepinephrine 伪 2 receptor on norepinephrine receptor mediated nociceptive synaptic transmission in spinal cord, and to reveal its possible molecular mechanism. Methods: the effects of norepinephrine 伪 2 receptor agonist on NMDA receptor in the spinal dorsal horn of mice with inflammatory pain were investigated by means of behavioral measurement, patch clamp electrophysiological recording and Western blot. Results Spinal cord sections were prepared 24 hours after injection of complete Freund's adjuvant CFAs into the posterior plantar of mice. The results of whole cell recording on lamellar neurons of dorsal horn II showed that the inflammatory pain model was made 24 hours later. Clonidine, a norepinephrine 伪 2 receptor agonist, significantly reduced the amplitude of the excitatory postsynaptic current (NMDAR-EPSCs) mediated by NMDA receptor in inflammatory pain mice via a2A subtype receptor. There was no significant change in Clonidine before extracellular perfusion, suggesting that the inhibitory effect of Clonidine on NMDAR-EPSCs in mice with inflammatory pain occurred in the postsynaptic membrane of MCA3). The G 伪 inhibitor GDP- 尾 -S- was introduced into the cells via recording electrode, which could significantly block the inhibitory effect of Clonidine on NMDAR-EPSCs. The direct inhibition of cAMP dependent protein kinase (PKA) can also mimic the inhibitory effect of Clonidine on NMDAR-EPSCs, suggesting that the whole experiment shows that: Clonidine may take effect by "GI protein / PKA signaling pathway". Intrathecal injection of Clonidine can specifically reduce the content of NMDA receptor (GluN2B receptor) containing GluN2B subunit in synapses. The results of in vitro electrophysiological records further prove that ifenprodil, a selective inhibitor of GluN2B receptor, can not only reduce inflammatory pain in mice. NMDA receptor mediated synaptic transmission, Moreover, the synaptic inhibitory effect of Clonidine was completely saturated, suggesting that norepinephrine 伪 2 receptor may play an analgesic effect by selectively down-regulating the synaptic function of GluN2B receptor. Conclusion: norepinephrine 伪 2 receptor may pass through the signal pathway of GI protein / PKA / GluN2B receptor, inhibit the pain synaptic transmission mediated by NMDA receptor in the spinal dorsal horn of inflammatory pain mice, and relieve chronic inflammatory pain symptoms.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965

【参考文献】