本文選題：脊髓背角　切入點(diǎn)：GABA能去抑制　出處：《蘭州大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：外周組織損傷造成的脊髓背角γ-氨基丁酸(γ-aminobutyric acid; GABA)能去抑制會激活cAMP依賴性蛋白激酶(cAMP-dependent protein kinases；PKA)；活化的PKA通過磷酸化61kD的紋狀體富集的蛋白酪氨酸磷酸酶(61kDStriatal-enriched protein tyrosine phosphatase; STEP61),能夠打斷STEP61與其底物Src家族酪氨酸激酶Fyn (Src family tyrosine kinase Fyn)、細(xì)胞外調(diào)節(jié)蛋白激酶1和2(Extracellular regulated protein kinases1and2; ERK1/2)之間的相互作用,引發(fā)細(xì)胞內(nèi)的多種信號通路。本研究的目標(biāo),在于探討GABA能抑制作用的恢復(fù),是否能夠通過增強(qiáng)STEP61的活性,有效緩解慢性炎性疼痛癥狀。 方法：本研究制備完全佛氏佐劑(Complete Freund's Adjuvant; CFA)慢性炎性疼痛模型；利用行為學(xué)檢測、免疫印跡、免疫沉淀、免疫共沉淀以及免疫組織化學(xué)等實(shí)驗(yàn)方法,深入探究了STEP61在GABA能去抑制惡化慢性炎性疼痛中的作用及其分子機(jī)制。 結(jié)果：(1)鞘內(nèi)注射重組腺病毒載體,能夠使脊髓背角神經(jīng)元時間依賴性地表達(dá)綠色熒光蛋白(Green Fluorescent Protein; GFP)標(biāo)記的外源性STEP61;(2)正常小鼠的脊髓背角表達(dá)無催化活性的STEP61突變體---STEP61(C472S),不僅會誘發(fā)明顯的痛覺超敏,而且會完全飽和(occlude) GABAA受體拮抗劑荷包牡丹堿(bicuculline;0.1μg)的致痛效應(yīng)；(3)荷包牡丹堿的重要作用,在于打斷STEP61與其底物Fyn、ERK1/2之間的分子結(jié)合,而這一作用可被N-甲基-D-天冬氨酸(N-Methyl-D-Aspartate; NMDA)受體拮抗劑D-APV (100μM)、高濃度Mg2+(10mM)完全阻斷,而Na+通道阻斷劑利多卡因(lidocaine;500μM)卻無此效應(yīng),提示：GABA能去抑制,可能通過增強(qiáng)NMDA受體的自發(fā)性突觸活動而干擾STEP61復(fù)合物的形成；(4)CFA能夠通過GABA能去抑制,激活脊髓背角PKA,破壞STEP61與Fyn、ERK1/2之間的相互作用；(5)炎性疼痛小鼠的鞘內(nèi)注射GABAA受體激動劑毒蠅蕈(muscimol;0.1μg),能夠恢復(fù)STEP61與其底物的結(jié)合,抑制Fyn以及ERK1/2的磷酸化；(6)為恢復(fù)STEP61的功能,本實(shí)驗(yàn)使脊髓背角過量表達(dá)外源性的、野生型STEP61---STEP61(WT),發(fā)現(xiàn)：STEP61(WT)能夠阻斷CFA誘發(fā)的Fyn、ERK1/2的異常磷酸化；(7)STEP61(WT)能夠同時阻斷CFA誘發(fā)的NMDA受體NR2B亞基第1472位酪氨酸(Try1472)的磷酸化,降低NR2B受體的突觸含量；(8)更為重要的是：過表達(dá)STEP61(WT)能夠有效抑制慢性炎性疼痛的誘導(dǎo)和持久維持。 結(jié)論：外周組織損傷通過脊髓背角GABA能去抑制,干擾STEP61對Fyn、ERK1/2的抑制性控制,誘發(fā)NMDA受體功能亢進(jìn)和痛覺超敏的形成；而直接表達(dá)外源性STEP61(WT)能夠有效緩解慢性炎性疼痛癥狀。
[Abstract]:Objective: 緯 -aminobutyric acid (GABA) induced by peripheral tissue injury can inhibit the activation of cAMP dependent protein kinase cAMP-dependent protein kinasesPKA; activated PKA is enriched with tyrosine phosphatase 61kDS triated-enriched protein tyrosine via phosphorylated 61kD striatum. The interaction between STEP61 and its substrate, Fyn family tyrosine kinase Fynn, extracellular regulated protein kines1 and 2; ERK1 / 2) was interrupted. The aim of this study is to investigate whether GABA can effectively relieve chronic inflammatory pain by enhancing the activity of STEP61. Methods: in this study, the complete Freund's adjuvant complete Freund's Adjuvant (CFAs) chronic inflammatory pain model was established, and the methods of behavioral detection, immunoblotting, immunoprecipitation, immunoprecipitation and immunohistochemistry were used. The role of STEP61 in GABA inhibition of chronic inflammatory pain and its molecular mechanism were investigated. Results the recombinant adenovirus vector was injected intrathecal. The STEP61 mutant STEP61C472SX, which can express green Fluorescent protein (GFP) labeled exogenous STEP61m-2) in spinal dorsal horn neurons in a time-dependent manner, not only induces obvious hyperalgesia, but also has no catalytic activity in the spinal dorsal horn of normal mice. The pain-inducing effect of bicuculline (0.1 渭 g), a completely saturated GABAA receptor antagonist, is important to interrupt the molecular binding between STEP61 and its substrate, Fynn ERK 1 / 2. This effect was completely blocked by N-Methyl-D-Aspartate; NMDA-receptor antagonist D-APV (100 渭 m), high concentration of Mg2 (10mm), but not by Na channel blocker lidocaineine (500 渭 M), suggesting that BGABA can be inhibited. It may interfere with the formation of STEP61 complex by enhancing spontaneous synaptic activity of NMDA receptor. Activation of PKA in the dorsal horn of spinal cord, disrupting the interaction between STEP61 and Fynn ERK 1 / 2) Intrathecal injection of GABAA receptor agonist muscimolor 0.1 渭 g / g in mice with inflammatory pain could restore the binding of STEP61 to its substrate and inhibit the phosphorylation of Fyn and ERK1/2) in order to restore the function of STEP61. In this study, the spinal dorsal horn overexpressed exogenous, wild-type STEP61WTT. It was found that CFA induced abnormal phosphorylation of Fynnberg ERK1 / 2 was blocked by W STEP61WTT), and the phosphorylation of NMDA receptor NR2B subunit 1472 tyrosine receptor NR2B subunit 1472 (Try1472) induced by CFA was also blocked in the dorsal horn of spinal cord. It is more important to reduce the synaptic content of NR2B receptor: overexpression of STEP61WT) can effectively inhibit the induction and sustained maintenance of chronic inflammatory pain. Conclusion: peripheral tissue injury can be inhibited by spinal dorsal horn GABA, interfere with the inhibitory control of STEP61 on Fynn ERK1 / 2, induce hyperfunction of NMDA receptor and the formation of pain hypersensitivity, while direct expression of exogenous STEP61WTcan effectively relieve 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】：2014
【分類號】：R965

【共引文獻(xiàn)】

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