角质细胞NMDA受体在复杂性区域疼痛综合征痛觉过敏中的作用研究
发布时间：2018-08-09 10:01
【摘要】：背景:复杂性区域疼痛综合征(complex regional pain syndrome,CRPS)是一种以痛觉过敏为主要表现的慢性、难治性临床综合征,严重危害患者生活质量。既往研究表明,角质细胞激活在外周疼痛传导中发挥重要作用,其上的α-氨基-3-羟基-5-甲基-4-异恶挫丙酸(α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid,AMPA)受体可参与带状疱疹后神经痛的机制。与AMPA受体同类的N-甲基-D-天冬氨酸(N-methyl-D-aspartic acidreceptor,NMDA)受体在角质细胞上也有表达,但其在痛觉过敏中的作用尚未明确。本研究拟探究角质细胞NMDA受体在CRPS痛觉过敏中的作用,重点观察其对促炎性细胞因子释放、疼痛传导和中枢胶质细胞激活的影响。方法:①动物分组与干预:本研究采用CRPS公认的模型——大鼠慢性缺血后疼痛(chronic post-ischemia pain,CPIP)模型,以 NMDA 受体激动剂 NMDA、拮抗剂MK801皮下注射的干预方法验证该受体作用。首先将雄性斯普拉格-道利(Sprague-Dawley,SD)大鼠分为急性期观察组与慢性期观察组:急性期观察组于建模前3日开始连续给药至建模日,慢性期观察组于建模后7日开始连续给药至第14日。急、慢性期观察组均细分为CPIP组与假手术组:CPIP组以内径0.65 cm的O型环于踝关节处勒紧右后肢,3小时后取下;假手术组以剪断的环做相同处理。CPIP组继续细分为3组,分别予NMDA(1 mM)、MK801(1 mM)和生理盐水100 μl/日。②行为学和生理学测定:急性观察组于建模后第6、12、18、24小时,慢性观察组于建模后第2、6、10、14日测定机械痛阈(von-Frey法)、热痛阈(热板法)和皮温(红外线温度计)。③取材:急、慢性观察组分别于建模后24小时和14日取大鼠手术侧皮肤及L2—L4脊髓。④Western Blot:测定皮肤组织NMDA受体固有亚基NR1表达水平,以及皮肤和脊髓中肿瘤坏死因子(tumor necrosis factor-α,TNF-α)和白介素-Iβ(interleukin-1 β,IL-1 β)的释放水平。⑤免疫荧光染色:观察皮肤组织角质细胞标志物pan-keratin与NR1共表达情况、脊髓背角伤害刺激传入标志物c-fos、小胶质细胞标志物钙离子结合调节分子 1(ionized calcium binding adaptor molecule 1,Iba-1)和星形胶质细胞标志物神经胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)的表达情况。结果:①行为与生理学:急性期和慢性期机械痛阈和热痛阈比较:CPIP+NMDA组CPIP+生理盐水组CPIP+MK801组假手术组;仅在急性期CPIP3组皮温均高于假手术组,但3组间无显著差异;②皮肤免疫荧光和Western Blot:在急、慢性期,角质细胞上NMDA受体的表达均显著升高。急性期TNF-a和IL-1 β释放量组间比较:CPIP+ NMDA组CPIP+生理盐水组CPIP+MK801组或假手术组,慢性期各组间无显著差异。③脊髓免疫荧光和Western Blot:CPIP+生理盐水组较假手术组急、慢性期均见脊髓背角c-fos表达提高,仅在慢性期见Ibal和GFAP表达升高,三者的表达在CPIP+MK801组中均被抑制。慢性期可见脊髓TNF-a和IL-1 β释放量组间比较:CPIP+ NMDA组CPIP+生理盐水组CPIP+MK801组或假手术组,而急性期无明显变化。结论:①CRPS急性期以皮肤的炎症反应为主,慢性期以中枢敏化为主;②CRPS急、慢性期均有角质细胞NMDA受体表达上调;③CRPS急性期角质细胞NMDA受体调控皮肤促炎性细胞因子的释放和疼痛传导;④CRPS慢性期角质细胞NMDA受体参与调控脊髓背角胶质细胞激活和促炎性细胞因子的释放。
[Abstract]:Background: complex regional pain syndrome (CRPS) is a chronic, refractory clinical syndrome characterized by hyperalgesia, which seriously endangering the patient's quality of life. Previous studies have shown that keratinocyte activation plays an important role in peripheral pain conduction, the alpha amino -3- hydroxyl -5- methyl -4- on it. The receptor of -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) can participate in the mechanism of post herpes zoster neuralgia. The N- methyl -D- aspartate (N-methyl-D-aspartic acidreceptor, NMDA) receptor, similar to AMPA receptor, also appears on the keratinocytes, but its role in hyperalgesia is not clear. Explore the role of keratinocyte NMDA receptor in CRPS hyperalgesia, focusing on its effects on proinflammatory cytokine release, pain conduction and central glial cell activation. Methods: (1) animal groups and interventions: This study uses a CRPS recognized model of chronic ischemic post ischemic pain (chronic post-ischemia pain, CPIP) model in rats The receptor action of NMDA receptor agonist NMDA and antagonist MK801 was tested subcutaneously. First, the male Sprague Dawley (Sprague-Dawley, SD) rats were divided into the acute phase observation group and the chronic phase observation group: the acute phase observation group began the continuous administration to the modeling day 3 days before the modeling, and the chronic phase observation group was 7 days after the modeling. The group of acute and chronic phase observation group were divided into group CPIP and sham operation group: group CPIP was subdivided into group CPIP and sham operation group: group CPIP with type O ring with inner diameter of the ankle in the ankle joint tightened right hind limbs, 3 hours after 3 hours, and the sham operation group continued to subdivide into 3 groups with the same treatment of clipping ring as.CPIP group, respectively, NMDA (1 mM), MK801 (1 mM) and physiological saline 100 um l/ day respectively. Behavioral and physiological measurements: the acute observation group was established at 6,12,18,24 hours after modeling, and the chronic observation group measured the mechanical pain threshold (von-Frey), the thermal pain threshold (hot plate method) and the skin temperature (infrared thermometer) on the 2,6,10,14 day after modeling. 3. The acute, chronic observation groups were separated from the operating side of the rat's skin and the L2 L4 ridge 24 hours and 14 days after the modeling. Western Blot: determination of the expression level of NMDA receptor inherent subunit NR1 in skin tissue and the release level of tumor necrosis factor (tumor necrosis factor- alpha, TNF- a) and interleukin -I beta (interleukin-1 beta, IL-1 beta) in skin and spinal cord. Conditions of spinal dorsal horn injury stimulation of the afferent marker c-fos, microglia marker calcium binding regulator 1 (ionized calcium binding adaptor molecule 1, Iba-1) and astroglia marker neuroglia fibrillary acidic protein (glial fibrillary acidic protein, GFAP) expression. Results: (1) behavioral and physiological: acute The mechanical pain threshold and the thermal pain threshold of the sexual and chronic phase were compared: group CPIP+MK801 of group CPIP+NMDA CPIP+ saline group CPIP+MK801 sham operation group; only in acute phase CPIP3 group the skin temperature was higher than that of sham operation group, but there was no significant difference between the 3 groups, and the expression of NMDA receptor on the skin immunofluorescence and Western Blot: increased significantly in acute, chronic stage, and on the acute stage of TN. The comparison of F-a and IL-1 beta release groups: there was no significant difference between the CPIP+MK801 group and the sham operation group in the CPIP+ physiological saline group of CPIP+ NMDA group, and there was no significant difference between the groups in the chronic phase. (3) the spinal cord immunofluorescence and the Western Blot:CPIP+ physiological saline group were more acute than the sham group, and the chronic phase of the spinal cord was higher in c-fos expression in the dorsal horn of the spinal cord, and the expression of Ibal and GFAP increased in the chronic phase only, three The expression in the CPIP+MK801 group was suppressed in the chronic phase of the group of TNF-a and IL-1 beta release in the CPIP+ NMDA group, the CPIP+ physiological saline group CPIP+MK801 group or the sham operation group, but there was no obvious change in the acute phase. Conclusion: (1) the acute phase of the CRPS is mainly caused by the inflammatory reaction of the skin, and the chronic phase is mainly central sensitization; (2) CRPS acute and chronic period The expression of keratinocyte NMDA receptor is up-regulated; (3) NMDA receptors in acute phase CRPS keratinocytes regulate the release and pain conduction of cutaneous proinflammatory cytokines; (4) the NMDA receptor of CRPS chronic phase keratinocytes is involved in the regulation of the activation of glial cells in the dorsal horn of the spinal cord and the release of proinflammatory cytokines.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R402
[Abstract]:Background: complex regional pain syndrome (CRPS) is a chronic, refractory clinical syndrome characterized by hyperalgesia, which seriously endangering the patient's quality of life. Previous studies have shown that keratinocyte activation plays an important role in peripheral pain conduction, the alpha amino -3- hydroxyl -5- methyl -4- on it. The receptor of -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) can participate in the mechanism of post herpes zoster neuralgia. The N- methyl -D- aspartate (N-methyl-D-aspartic acidreceptor, NMDA) receptor, similar to AMPA receptor, also appears on the keratinocytes, but its role in hyperalgesia is not clear. Explore the role of keratinocyte NMDA receptor in CRPS hyperalgesia, focusing on its effects on proinflammatory cytokine release, pain conduction and central glial cell activation. Methods: (1) animal groups and interventions: This study uses a CRPS recognized model of chronic ischemic post ischemic pain (chronic post-ischemia pain, CPIP) model in rats The receptor action of NMDA receptor agonist NMDA and antagonist MK801 was tested subcutaneously. First, the male Sprague Dawley (Sprague-Dawley, SD) rats were divided into the acute phase observation group and the chronic phase observation group: the acute phase observation group began the continuous administration to the modeling day 3 days before the modeling, and the chronic phase observation group was 7 days after the modeling. The group of acute and chronic phase observation group were divided into group CPIP and sham operation group: group CPIP was subdivided into group CPIP and sham operation group: group CPIP with type O ring with inner diameter of the ankle in the ankle joint tightened right hind limbs, 3 hours after 3 hours, and the sham operation group continued to subdivide into 3 groups with the same treatment of clipping ring as.CPIP group, respectively, NMDA (1 mM), MK801 (1 mM) and physiological saline 100 um l/ day respectively. Behavioral and physiological measurements: the acute observation group was established at 6,12,18,24 hours after modeling, and the chronic observation group measured the mechanical pain threshold (von-Frey), the thermal pain threshold (hot plate method) and the skin temperature (infrared thermometer) on the 2,6,10,14 day after modeling. 3. The acute, chronic observation groups were separated from the operating side of the rat's skin and the L2 L4 ridge 24 hours and 14 days after the modeling. Western Blot: determination of the expression level of NMDA receptor inherent subunit NR1 in skin tissue and the release level of tumor necrosis factor (tumor necrosis factor- alpha, TNF- a) and interleukin -I beta (interleukin-1 beta, IL-1 beta) in skin and spinal cord. Conditions of spinal dorsal horn injury stimulation of the afferent marker c-fos, microglia marker calcium binding regulator 1 (ionized calcium binding adaptor molecule 1, Iba-1) and astroglia marker neuroglia fibrillary acidic protein (glial fibrillary acidic protein, GFAP) expression. Results: (1) behavioral and physiological: acute The mechanical pain threshold and the thermal pain threshold of the sexual and chronic phase were compared: group CPIP+MK801 of group CPIP+NMDA CPIP+ saline group CPIP+MK801 sham operation group; only in acute phase CPIP3 group the skin temperature was higher than that of sham operation group, but there was no significant difference between the 3 groups, and the expression of NMDA receptor on the skin immunofluorescence and Western Blot: increased significantly in acute, chronic stage, and on the acute stage of TN. The comparison of F-a and IL-1 beta release groups: there was no significant difference between the CPIP+MK801 group and the sham operation group in the CPIP+ physiological saline group of CPIP+ NMDA group, and there was no significant difference between the groups in the chronic phase. (3) the spinal cord immunofluorescence and the Western Blot:CPIP+ physiological saline group were more acute than the sham group, and the chronic phase of the spinal cord was higher in c-fos expression in the dorsal horn of the spinal cord, and the expression of Ibal and GFAP increased in the chronic phase only, three The expression in the CPIP+MK801 group was suppressed in the chronic phase of the group of TNF-a and IL-1 beta release in the CPIP+ NMDA group, the CPIP+ physiological saline group CPIP+MK801 group or the sham operation group, but there was no obvious change in the acute phase. Conclusion: (1) the acute phase of the CRPS is mainly caused by the inflammatory reaction of the skin, and the chronic phase is mainly central sensitization; (2) CRPS acute and chronic period The expression of keratinocyte NMDA receptor is up-regulated; (3) NMDA receptors in acute phase CRPS keratinocytes regulate the release and pain conduction of cutaneous proinflammatory cytokines; (4) the NMDA receptor of CRPS chronic phase keratinocytes is involved in the regulation of the activation of glial cells in the dorsal horn of the spinal cord and the release of proinflammatory cytokines.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R402
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