角質(zhì)細胞NMDA受體在復雜性區(qū)域疼痛綜合征痛覺過敏中的作用研究
發(fā)布時間：2018-08-09 10:01
【摘要】：背景:復雜性區(qū)域疼痛綜合征(complex regional pain syndrome,CRPS)是一種以痛覺過敏為主要表現(xiàn)的慢性、難治性臨床綜合征,嚴重危害患者生活質(zhì)量。既往研究表明,角質(zhì)細胞激活在外周疼痛傳導中發(fā)揮重要作用,其上的α-氨基-3-羥基-5-甲基-4-異惡挫丙酸(α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid,AMPA)受體可參與帶狀皰疹后神經(jīng)痛的機制。與AMPA受體同類的N-甲基-D-天冬氨酸(N-methyl-D-aspartic acidreceptor,NMDA)受體在角質(zhì)細胞上也有表達,但其在痛覺過敏中的作用尚未明確。本研究擬探究角質(zhì)細胞NMDA受體在CRPS痛覺過敏中的作用,重點觀察其對促炎性細胞因子釋放、疼痛傳導和中樞膠質(zhì)細胞激活的影響。方法:①動物分組與干預:本研究采用CRPS公認的模型——大鼠慢性缺血后疼痛(chronic post-ischemia pain,CPIP)模型,以 NMDA 受體激動劑 NMDA、拮抗劑MK801皮下注射的干預方法驗證該受體作用。首先將雄性斯普拉格-道利(Sprague-Dawley,SD)大鼠分為急性期觀察組與慢性期觀察組:急性期觀察組于建模前3日開始連續(xù)給藥至建模日,慢性期觀察組于建模后7日開始連續(xù)給藥至第14日。急、慢性期觀察組均細分為CPIP組與假手術(shù)組:CPIP組以內(nèi)徑0.65 cm的O型環(huán)于踝關節(jié)處勒緊右后肢,3小時后取下;假手術(shù)組以剪斷的環(huán)做相同處理。CPIP組繼續(xù)細分為3組,分別予NMDA(1 mM)、MK801(1 mM)和生理鹽水100 μl/日。②行為學和生理學測定:急性觀察組于建模后第6、12、18、24小時,慢性觀察組于建模后第2、6、10、14日測定機械痛閾(von-Frey法)、熱痛閾(熱板法)和皮溫(紅外線溫度計)。③取材:急、慢性觀察組分別于建模后24小時和14日取大鼠手術(shù)側(cè)皮膚及L2—L4脊髓。④Western Blot:測定皮膚組織NMDA受體固有亞基NR1表達水平,以及皮膚和脊髓中腫瘤壞死因子(tumor necrosis factor-α,TNF-α)和白介素-Iβ(interleukin-1 β,IL-1 β)的釋放水平。⑤免疫熒光染色:觀察皮膚組織角質(zhì)細胞標志物pan-keratin與NR1共表達情況、脊髓背角傷害刺激傳入標志物c-fos、小膠質(zhì)細胞標志物鈣離子結(jié)合調(diào)節(jié)分子 1(ionized calcium binding adaptor molecule 1,Iba-1)和星形膠質(zhì)細胞標志物神經(jīng)膠質(zhì)纖維酸性蛋白(glial fibrillary acidic protein,GFAP)的表達情況。結(jié)果:①行為與生理學:急性期和慢性期機械痛閾和熱痛閾比較:CPIP+NMDA組CPIP+生理鹽水組CPIP+MK801組假手術(shù)組;僅在急性期CPIP3組皮溫均高于假手術(shù)組,但3組間無顯著差異;②皮膚免疫熒光和Western Blot:在急、慢性期,角質(zhì)細胞上NMDA受體的表達均顯著升高。急性期TNF-a和IL-1 β釋放量組間比較:CPIP+ NMDA組CPIP+生理鹽水組CPIP+MK801組或假手術(shù)組,慢性期各組間無顯著差異。③脊髓免疫熒光和Western Blot:CPIP+生理鹽水組較假手術(shù)組急、慢性期均見脊髓背角c-fos表達提高,僅在慢性期見Ibal和GFAP表達升高,三者的表達在CPIP+MK801組中均被抑制。慢性期可見脊髓TNF-a和IL-1 β釋放量組間比較:CPIP+ NMDA組CPIP+生理鹽水組CPIP+MK801組或假手術(shù)組,而急性期無明顯變化。結(jié)論:①CRPS急性期以皮膚的炎癥反應為主,慢性期以中樞敏化為主;②CRPS急、慢性期均有角質(zhì)細胞NMDA受體表達上調(diào);③CRPS急性期角質(zhì)細胞NMDA受體調(diào)控皮膚促炎性細胞因子的釋放和疼痛傳導;④CRPS慢性期角質(zhì)細胞NMDA受體參與調(diào)控脊髓背角膠質(zhì)細胞激活和促炎性細胞因子的釋放。
[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.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2017
【分類號】：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.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R402
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