神经病理性疼痛节律及其机制研究

发布时间：2018-05-24 09:44

本文选题：CREB + CRTC1　；参考：《南京大学》2015年博士论文

【摘要】：研究背景:大量临床调查显示,慢性疼痛普遍存在昼夜节律性改变,且大多表现出夜间的疼痛高峰。昼夜节律依赖内在的生物钟发挥作用,生物钟基因通过基因转录及编码的蛋白质组成钟振荡器,维持内源性生物钟的运作。下丘脑视交叉上核作为生物钟中枢控制器,可调控其他节律振荡器来协调机体的各种生理活动和行为过程。参与疼痛信号调制的脊髓背角钟基因可关联至痛行为的节律改变。脊髓背角伤害性感受神经元的敏化过程被认为参与到疼痛的各环节,NR2B受体在中枢敏化的诱发和维持中至关重要,并可作用于下游信号分子CRTC1及CREB,CRTC1作为CREB的共激活分子可级联放大CREB的磷酸化表达,参与疼痛的形成和维持。近期研究亦提示NR2B-CRTC1-CREB信号通路亦与视交叉上核所介导的昼夜节律有直接关联,其与神经病理性疼痛节律性变化的机制联系值得探讨。研究目的:1、探索正常昼夜节律下脊髓背角与中枢控制器的钟基因表达关联性,观察神经病理性痛下脊髓背角钟基因节律及"睡眠-觉醒"节律的改变;2、明确神经病理性疼痛痛觉敏化行为的节律改变;3、揭示NR2B-CRTC1-CREB信号通路在谷氨酸诱导的体外原代培养小鼠脊髓神经元中的机制联系;4、探讨以NR2B-CRTC1-CREB信号通路与神经病理性疼痛节律的机制关联为切入点的临床慢性疼痛诊疗新思路。研究方法:建立小鼠及大鼠坐骨神经结扎模型(Chronic Constriction Injury,CCI)模拟神经病理性疼痛;谷氨酸刺激原代培养小鼠脊髓神经元体外模拟疼痛发生,并激活CREB信号通路;制备CREB及CRTC1干扰腺病毒载体,在疼痛稳定期昼夜交替点使用其进行干预,观察疼痛行为学及NR2B-CRTC1-CREB信号通路改变;机械缩足阈值及热辐射潜伏期测定评估小鼠及大鼠痛觉敏化行为;Minimitter全程实时监测小鼠"睡眠-觉醒"节律相关的粗大运动改变;CCK-8检测药物干预对神经元的杀伤情况;通过免疫荧光、Western Blot和实时荧光定量PCR等分子生物学手段探讨相关的分子机制改变。研究结果:1、正常昼夜节律下小鼠脊髓背角及外周血单个核细胞的生物钟基因Cry1、Per2、Bmal1及Clock的mRNA表达与位于下丘脑视交叉上核区的中枢节律振荡器呈现相对同步性,坐骨神经结扎模型小鼠痛觉稳定期脊髓背角钟基因的表达具有明显节律性振荡。2、坐骨神经结扎模型小鼠"睡眠-觉醒"节律未受影响;相对睡眠期粗大运动较假手术组显著减少。3、坐骨神经痛动物机械缩足阈值与热辐射潜伏期呈现夜高昼低的节律性改变。4、体外原代培养的小鼠脊髓神经元在谷氨酸最适浓度100μM刺激下,可磷酸化NR2B及CREB;使用CRTC1及CREB干扰腺病毒载体可明显抑制相关基因的mRNA表达。5、脊髓背角NR2B、CRTC1及CREB的mRNA、活化状态的免疫荧光及蛋白水平改变均与坐骨神经结扎小鼠疼痛节律呈高度一致。6、抑制CRTC1及CREB可明显减少痛敏行为发生;在疼痛阈值低谷前的昼夜交替点给予干预可更有效调节疼痛节律;NR2B-CRTC1-CREB反馈性调节信号通路与痛行为改善具明显相关性。研究结论:1、小鼠脊髓背角生物钟基因表达的昼夜节律与中枢振荡器呈现相对同步性,神经病理性疼痛下脊髓背角钟基因可节律性振荡。2、神经病理性疼痛下可呈现以夜间痛为特点的疼痛节律,"睡眠-觉醒"节律未受影响。3、调控NR2B-CRTC1-CREB信号通路可直接影响神经病理性疼痛节律,并改善痛觉敏化。
[Abstract]:Background: a large number of clinical studies have shown that chronic pain is generally circadian rhythmic changes, and most of them show nocturnal peak of pain. The circadian rhythm relies on the internal clock to play a role. The clock gene makes the clock oscillator through gene transcription and encoded protein to maintain the operation of endogenous circadian clock. The hypothalamic optic cross has been maintained. The upper nucleus, as the central controller of the biological clock, regulates the other rhythmic oscillators to coordinate the physiological and behavioral processes of the body. The spinal dorsal horn clock genes involved in the pain signal modulation can be associated with the rhythmic changes in the pain behavior. The sensitization of the nociceptive neurons in the dorsal horn of the spinal cord is considered to be involved in all aspects of the pain, and NR2B is affected. Body plays an important role in the induction and maintenance of central sensitization, and can act on the downstream signal molecules CRTC1 and CREB. As a co activator of CREB, CRTC1 can cascade to amplify the phosphorylation of CREB and participate in the formation and maintenance of pain. Recent studies also suggest that the NR2B-CRTC1-CREB signaling pathway is also directly related to the circadian rhythm mediated by the suprachiasmatic nucleus. The relationship between the neuropathic pain and the rhythmic changes of neuropathic pain should be discussed. 1. To explore the correlation between the dorsal horn of the spinal cord and the central controller's clock gene expression under normal circadian rhythm, to observe the changes of the genetic rhythm of the spinal dorsal horn and the "sleep awakening" rhythm under the neuropathic pain, and 2, to clarify the neuropathic pain. The rhythmic changes of pain sensitized behavior; 3, reveal the mechanism of NR2B-CRTC1-CREB signaling pathway in the primary cultured mouse spinal cord neurons induced by glutamic acid; 4, explore a new approach to clinical chronic pain diagnosis and treatment with the mechanism of NR2B-CRTC1-CREB signaling pathway and the mechanism of neuropathic pain rhythm. Chronic Constriction Injury (CCI) simulated neuropathic pain in mice and rats; glutamic acid stimulated the primary cultured mouse spinal neurons to simulate pain in vitro, and activated the CREB signaling pathway; the CREB and CRTC1 interfering adenovirus vectors were prepared and used to intervene in the day and night alternation points of the pain stable period. Observe pain behavior and change of NR2B-CRTC1-CREB signal pathway; measure the threshold of mechanical contraction and latent period of heat radiation to evaluate the pain sensitization behavior of mice and rats; Minimitter monitoring the gross motor change related to the "sleep awakening" rhythm of mice in real time; CCK-8 detection of the killing of neurons by drug preconditioning; by immunofluorescence, Wester Molecular biological means such as n Blot and real time fluorescence quantitative PCR are discussed. The results are as follows: 1, the mRNA expression of the clock gene Cry1, Per2, Bmal1 and Clock in the spinal dorsal horn and peripheral blood mononuclear cells of mice under normal circadian rhythm is relative to the central rhythmic oscillator located in the supramoral nucleus of the hypothalamus. The expression of the dorsal horn clock gene in the spinal cord of the mice with the sciatic nerve ligature has an obvious rhythmic oscillation.2, and the sleep awakening rhythm of the sciatic nerve ligature model mice is not affected; the relative sleep period is significantly reduced by.3 in the relative sleep period than in the sham operation group, and the threshold of the mechanical contraction of the sciatic deity pain and the latent period of the thermal radiation exposure are presented. The rhythmic change of night high day low.4, the primary cultured mouse spinal cord neurons were phosphorylated NR2B and CREB under the optimum concentration of glutamic acid 100 micron M. CRTC1 and CREB interfering adenovirus vector could obviously inhibit the mRNA expression.5 of the related genes, NR2B, CRTC1 and CREB mRNA, activated immunofluorescence and protein water in the spinal dorsal horn. The pain rhythm of the sciatic nerve ligation mice was highly consistent with.6, and the inhibition of CRTC1 and CREB could significantly reduce the occurrence of pain sensitization; the intervention of day and night alternation points before the threshold of the pain threshold could be more effective in regulating the pain rhythm; the NR2B-CRTC1-CREB feedback regulation signal pathway was significantly correlated with the improvement of pain behavior. 1 The circadian rhythm of the circadian clock gene expression in the spinal dorsal horn of the mouse is relative synchrony with the central oscillator, and the spinal dorsal horn clock gene can oscillate rhythmically under neuropathic pain, and the pain rhythms characterized by nocturnal pain can be presented under neuropathic pain. The "sleep wake" rhythm is not affected by.3 and regulates the NR2B-CRTC1-CREB signal. Lu can directly affect the rhythm of neuropathic pain and improve pain sensitization.
【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R614

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