本文選題：去甲腎上腺素α2A受體 + C-末端Src激酶�。� 參考：《蘭州大學(xué)》2015年碩士論文

【摘要】：目的：去甲腎上腺素α2受體和抑制性Gi蛋白相偶聯(lián),通過Gi蛋白α亞基(Giα)和βγ亞基(Gβγ)觸發(fā)多種細(xì)胞內(nèi)信號轉(zhuǎn)導(dǎo)通路。α2受體包含多種亞型；其中,A型去甲腎上腺素α2受體(α2A受體)表達(dá)于脊髓背角、并在痛覺信息的調(diào)控中發(fā)揮著至關(guān)重要的作用。目前為止,絕大多數(shù)實(shí)驗(yàn)探討了Giα亞基誘發(fā)的cAMP信號通路與痛覺調(diào)制的關(guān)系；但Gβγ亞基的作用尚不清楚。本研究的目的,在于探討Gβγ通路在α2A受體抑制慢性炎性疼痛中的作用及其分子機(jī)制。方法：本研究給小鼠后足底皮下注射完全弗氏佐劑(Complete Freund's Adjuvant, CFA)、建立慢性炎性疼痛模型；構(gòu)建腺病毒表達(dá)載體,使脊髓背角表達(dá)Gβγ亞基的特異性阻斷劑PARKct(即：195個氨基酸組成的β-腎上腺素受體激酶-1的胞漿C-末端序列)；鞘內(nèi)注射a2A受體的選擇性激動劑Guanfacine,通過痛行為學(xué)測試、免疫印跡、膜片鉗電生理記錄、免疫沉淀等方法,探討了PARKct對 Guanfacine效應(yīng)的影響。結(jié)果：(1)鞘內(nèi)注射α2A受體激動劑Guanfacine (0.28μg),能夠有效緩解CFA誘發(fā)的熱痛覺過敏和機(jī)械性痛覺超敏,但這一作用可被PARKct拮抗；(2)α2A受體可能通過Gβγ亞基下調(diào)N-甲基-D-天冬氨酸(N-Methyl-D-Aspartate; NMD A)型谷氨酸受體的突觸表達(dá),因?yàn)棣翧RKct會阻斷Guanfacine對突觸小體膜結(jié)構(gòu)中NMDA受體GluNl/GluN2B亞基的抑制效應(yīng)；(3)免疫印跡實(shí)驗(yàn)顯示：α2A受體通過Gβγ亞基降低GluN2B亞基第1472位酪氨酸殘基(GluN2B-Tyrl472)的磷酸化水平,從而誘發(fā)包含GluN2B亞基的NMDA受體的內(nèi)陷；(4)免疫沉淀實(shí)驗(yàn)顯示：GluN2B亞基的催化激酶---Src,可能是α2A受體/Gβγ亞基的重要調(diào)控靶點(diǎn),因?yàn)椋篏uanfacine能夠降低Src的第418位酪氨酸殘基(Tyr418)的磷酸化、同時升高其529位酪氨酸殘基(Tyr529)的磷酸化,而預(yù)先表達(dá)βARKct則拮抗Guanfacine對Src活性的影響；(5)α2A受體/Gβγ亞基對Src的調(diào)節(jié)作用,可能與C-末端-Src激酶(CSK)有關(guān)；我們發(fā)現(xiàn)：βARKct能夠取消Guanfacine誘導(dǎo)的炎性疼痛小鼠脊髓背角CSK的突觸再分布；(6)為直接探討CSK的痛覺調(diào)控作用,我們構(gòu)建重組腺病毒載體,使脊髓表達(dá)無活性的CSK突變體CSK(K222R),結(jié)果發(fā)現(xiàn)：在正常動物,CSK(K222R)能夠升高GluN2B-Tyr1472的磷酸化水平；而在炎性疼痛動物,CSK(K222R)會阻斷Guanfacine對突觸NMDA受體的調(diào)節(jié)效應(yīng)；(7)尤為重要的是：通過重組腺病毒載體、使炎性疼痛動物的脊髓背角表達(dá)野生型CSK[CSK(WT)],能夠產(chǎn)生和Guanfacine相似的效應(yīng),即：降低GluNl/GluN2B亞基的突觸表達(dá)水平,逆轉(zhuǎn)GluN2B-Tyr1472的磷酸化,阻斷NMDA受體介導(dǎo)的痛覺突觸傳遞,有效緩解慢性炎性疼痛癥狀。結(jié)論：去甲腎上腺素α2A受體,能夠通過Gβγ/CSK信號通路,抑制Src對GluN2B的磷酸化、降低GluN2B的突觸表達(dá),這可能是α2A受體干預(yù)慢性炎性疼痛的一條重要途徑。
[Abstract]:Objective: to couple norepinephrine 偽 2 receptor with inhibitory GI protein, and trigger many intracellular signal transduction pathways via GI 偽 subunit (GI 偽) and 尾 緯 subunit (G 尾 緯). Type A norepinephrine 偽 2 receptor (偽 2A receptor) is expressed in the dorsal horn of spinal cord and plays an important role in the regulation of pain information. Up to now, most experiments have explored the relationship between camp signaling pathway induced by GI 偽 subunit and pain modulation, but the role of G 尾 緯 subunit is not clear. The purpose of this study was to investigate the role of G 尾 緯 pathway in 偽 2A receptor inhibiting chronic inflammatory pain and its molecular mechanism. Methods: in this study, a chronic inflammatory pain model was established by subcutaneous injection of complete FreundsAdjuvant (CFA) into the posterior plantar of mice, and adenovirus expression vector was constructed. PARKct, a specific inhibitor of G 尾 緯 subunit in spinal dorsal horn, was injected intrathecally with Guanfacine, a selective agonist of a2A receptor. The effects of PARKct on Guanfacine effect were investigated by patch clamp electrophysiological recording and immunoprecipitation. Results: (1) Intrathecal injection of 偽 2A receptor agonist Guanfacine (0. 28 渭 g),) could effectively relieve thermal hyperalgesia and mechanical hyperalgesia induced by CFA, but this effect could be antagonized by PARKct. (2) 偽 2A receptor may down-regulate the synaptic expression of N-Methyl-D-Aspartate (NMD A) glutamate receptor through G 尾 緯 subunit, because 尾 ARKct can block the inhibitory effect of Guanfacine on the NMDA receptor GluNl / GluN2B subunit in the membrane structure of synaptosome. (3) Western blot showed that 偽 2A receptor reduced the phosphorylation level of the 1472 tyrosine residue (GluN2B-Tyrl472) of GluN2B subunit through G 尾 緯 subunit, which induced the invagination of NMDA receptor containing GluN2B subunit. (4) Immunoprecipitation assay showed that the catalytic kinase -Src of the 1: GluN2B subunit may be an important regulatory target of 偽 2A receptor / G 尾 緯 subunit, because the tyrosine residue (Tyr418) phosphorylation of the 418th position of Src can be reduced by the weight Guanfacine. At the same time, the phosphorylation of Tyr529 tyrosine residue (Tyr529) was increased, and the effect of Guanfacine on SRC activity was antagonized by 尾 ARKct. (5) the regulation of SRC by 偽 2A receptor / G 尾 緯 subunit may be related to C-terminal -Src kinase (CSK). We found that 尾 ARKct could cancel the synaptic redistribution of CSK in the spinal dorsal horn of Guanfacine induced inflammatory pain mice. (6) in order to investigate the pain regulation of CSK directly, we constructed recombinant adenovirus vector. CSK mutant CSK (K222R), which made spinal cord expression inactive, was found that CSK (K222R) could increase the phosphorylation level of GluN2B-Tyr1472 in normal animals, while in inflammatory pain animals, CSK (K222R) blocked the regulatory effect of Guanfacine on synaptic NMDA receptor. (7) it is particularly important that the expression of wild type CSK [CSK (WT)] in the spinal dorsal horn of inflammatory pain animals can be induced by recombinant adenovirus vector, which can produce a similar effect to Guanfacine, that is, to reduce the synaptic expression level of GluNl / GluN2B subunit and reverse the phosphorylation of GluN2B-Tyr1472. Blocking NMDA receptor mediated nociceptive synaptic transmission effectively alleviates chronic inflammatory pain symptoms. Conclusion: norepinephrine 偽 2A receptor can inhibit Src phosphorylation of GluN2B and decrease the synaptic expression of GluN2B through G 尾 緯 -CSK signaling pathway, which may be an important pathway for 偽 2A receptor to interfere with chronic inflammatory pain.
【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】：2015
【分類號】：R402

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