脊髓背角STEP61抑制慢性炎性疼痛的分子机制

发布时间：2018-03-03 03:05

本文选题：脊髓背角　切入点：GABA能去抑制　出处：《兰州大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：外周组织损伤造成的脊髓背角γ-氨基丁酸(γ-aminobutyric acid; GABA)能去抑制会激活cAMP依赖性蛋白激酶(cAMP-dependent protein kinases；PKA)；活化的PKA通过磷酸化61kD的纹状体富集的蛋白酪氨酸磷酸酶(61kDStriatal-enriched protein tyrosine phosphatase; STEP61),能够打断STEP61与其底物Src家族酪氨酸激酶Fyn (Src family tyrosine kinase Fyn)、细胞外调节蛋白激酶1和2(Extracellular regulated protein kinases1and2; ERK1/2)之间的相互作用,引发细胞内的多种信号通路。本研究的目标,在于探讨GABA能抑制作用的恢复,是否能够通过增强STEP61的活性,有效缓解慢性炎性疼痛症状。方法：本研究制备完全佛氏佐剂(Complete Freund's Adjuvant; CFA)慢性炎性疼痛模型；利用行为学检测、免疫印迹、免疫沉淀、免疫共沉淀以及免疫组织化学等实验方法,深入探究了STEP61在GABA能去抑制恶化慢性炎性疼痛中的作用及其分子机制。结果：(1)鞘内注射重组腺病毒载体,能够使脊髓背角神经元时间依赖性地表达绿色荧光蛋白(Green Fluorescent Protein; GFP)标记的外源性STEP61;(2)正常小鼠的脊髓背角表达无催化活性的STEP61突变体---STEP61(C472S),不仅会诱发明显的痛觉超敏,而且会完全饱和(occlude) GABAA受体拮抗剂荷包牡丹碱(bicuculline;0.1μg)的致痛效应；(3)荷包牡丹碱的重要作用,在于打断STEP61与其底物Fyn、ERK1/2之间的分子结合,而这一作用可被N-甲基-D-天冬氨酸(N-Methyl-D-Aspartate; NMDA)受体拮抗剂D-APV (100μM)、高浓度Mg2+(10mM)完全阻断,而Na+通道阻断剂利多卡因(lidocaine;500μM)却无此效应,提示：GABA能去抑制,可能通过增强NMDA受体的自发性突触活动而干扰STEP61复合物的形成；(4)CFA能够通过GABA能去抑制,激活脊髓背角PKA,破坏STEP61与Fyn、ERK1/2之间的相互作用；(5)炎性疼痛小鼠的鞘内注射GABAA受体激动剂毒蝇蕈(muscimol;0.1μg),能够恢复STEP61与其底物的结合,抑制Fyn以及ERK1/2的磷酸化；(6)为恢复STEP61的功能,本实验使脊髓背角过量表达外源性的、野生型STEP61---STEP61(WT),发现：STEP61(WT)能够阻断CFA诱发的Fyn、ERK1/2的异常磷酸化；(7)STEP61(WT)能够同时阻断CFA诱发的NMDA受体NR2B亚基第1472位酪氨酸(Try1472)的磷酸化,降低NR2B受体的突触含量；(8)更为重要的是：过表达STEP61(WT)能够有效抑制慢性炎性疼痛的诱导和持久维持。结论：外周组织损伤通过脊髓背角GABA能去抑制,干扰STEP61对Fyn、ERK1/2的抑制性控制,诱发NMDA受体功能亢进和痛觉超敏的形成；而直接表达外源性STEP61(WT)能够有效缓解慢性炎性疼痛症状。
[Abstract]:Objective: 纬 -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.
【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

【共引文献】