本文選題：去甲腎上腺素α2受體 + NMDA受體��； 參考：《蘭州大學(xué)》2014年碩士論文

【摘要】：目的：去甲腎上腺素α2受體高度富集在脊髓背角。外周組織損傷發(fā)生時,鞘內(nèi)注射去甲腎上腺素α2受體激動劑可以有效地緩解病理性疼痛。但其確切的發(fā)生機制,目前尚不十分清楚。NMDA(N-甲基-D-天冬氨酸)型谷氨酸受體功能亢進(jìn),被認(rèn)為是誘發(fā)慢性病理性疼痛的核心機制之一。本課題的目標(biāo),在于探討去甲腎上腺素α2受體對脊髓NMDA受體介導(dǎo)的痛覺突觸傳遞的調(diào)節(jié)作用,并揭示其可能的分子機制。 方法：本研究運用行為學(xué)測定、膜片鉗電生理記錄和免疫蛋白印跡等方法,探討去甲腎上腺素α2受體激動劑對炎性疼痛小鼠脊髓背角NMDA受體的調(diào)節(jié)作用及其信號轉(zhuǎn)導(dǎo)通路。 結(jié)果：(1)小鼠后足底皮下注射完全弗氏佐劑(CFA)制備炎性疼痛模型,24h后制得脊髓切片,在背角II板層神經(jīng)元上的全細(xì)胞記錄結(jié)果顯示,去甲腎上腺素α2受體激動劑Clonidine通過a2A亞型受體,顯著地降低炎性疼痛小鼠NMDA受體介導(dǎo)的興奮性突觸后電流(NMDAR-EPSCs)的幅值；(2)記錄、并計算雙刺激比值(paired-pulse ratio; PPR;=2nd response/1st response)發(fā)現(xiàn),細(xì)胞外灌流Clonidine前、后,PPR并不發(fā)生明顯的變化,提示：Clonidine抑制炎性疼痛小鼠NMDAR-EPSCs的作用發(fā)生在突觸后膜；(3)通過記錄電極、向細(xì)胞內(nèi)導(dǎo)入Gα抑制劑GDP-β-S,可明顯阻斷Clonidine對NMDAR-EPSCs的抑制作用；而直接抑制cAMP依賴蛋白激酶(PKA),也可以模擬Clonidine對NMDAR-EPSCs的抑制效應(yīng),提示：Clonidine可能通過“Gi蛋白/PKA信號通路”起效：(4)整體實驗結(jié)果顯示,鞘內(nèi)注射Clonidine可特異性降低突觸中包含GluN2B亞基的NMDA受體(即GluN2B受體)的含量：(5)離體電生理記錄的結(jié)果進(jìn)一步證明,GluN2B受體選擇性抑制劑ifenprodil不僅能夠降低炎性疼痛小鼠NMDA受體介導(dǎo)的突觸傳遞,而且會完全飽和Clonidine的突觸抑制效應(yīng),提示：去甲腎上腺素α2受體可能通過選擇性下調(diào)GluN2B受體的突觸功能而發(fā)揮鎮(zhèn)痛作用。 結(jié)論：去甲腎上腺素α2受體可能通Gi蛋白/PKA/GluN2B受體信號通路,抑制炎性疼痛小鼠脊髓背角中NMDA受體介導(dǎo)的痛覺突觸傳遞,緩解慢性炎性疼痛癥狀。
[Abstract]:Objective: norepinephrine 偽 2 receptor is highly enriched in the dorsal horn of spinal cord. Intrathecal injection of norepinephrine 偽 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 偽 2 receptor on norepinephrine receptor mediated nociceptive synaptic transmission in spinal cord, and to reveal its possible molecular mechanism. Methods: the effects of norepinephrine 偽 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 偽 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 偽 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 偽 2 receptor may play an analgesic effect by selectively down-regulating the synaptic function of GluN2B receptor. Conclusion: norepinephrine 偽 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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R965

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本文編號：1981152