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目的:为了探讨氯离子转运体的表达及其功能在神经病理性疼痛的产生和维持中的作用。方法:建立大鼠坐骨神经压迫模型(chronic constriction injury,CCI);检测大鼠热刺激缩足反射潜伏期(thermal withdrawal latency,TWL);应用免疫组化、Western Blot技术检测钠-钾-氯共转运体1(sodium-potassium-chloride co-transorter 1,NKCC1)和钾-氯共转运体2(potassium-chloride co-transorter2,KCC2)在CCI模型背根神经节(dorsal root ganglion,DRG)神经元上表达的改变;应用氯离子荧光探针MQAE(N-Ethoxycarbonylmethyl-6-methoxyquinolinium bromide)检测DRG神经元氯离子浓度的变化。结果:(1)CCI组大鼠TWL较正常对照组明显缩短(P<0.01,n=10),至术后21天测试结束仍较明显(P<0.05,n=10)。假手术组与正常组差异无统计学意义。(2)正常情况下,NKCC1微弱表达于DRG大、中、小型神经元,KCC2未见表达。CCI模型术后第7天、14天、21天,NKCC1表达增加,KCC2未见表达。(3)CCI组大鼠术后第7天DRG、NKCC1阳性细胞计数、中型神经元显著增加(P<0.01,n=1000);术后第14天,中、小型神经元增加(P<0.01,n=1000);术后第21天,中型神经元增加(P<0.01,n=1000)。(4)CCI术后14、21天DRG神经元NKCC1蛋白表达显著增加。(5)CCI术后7、14天DRG神经元中氯离子浓度明显升高(P<0.05,n=1000)。结论:神经病理性疼痛发生时,NKCC1在DRG神经元表达异常增加,引起细胞内Cl-浓度升高,继而可能参与了介导初极感觉外周末梢去极化(primary afferent depolarization,PAD)或/和诱发初极感觉中枢末梢端的背根反射(dorsal root reflexes,DRRs)引发痛觉过敏和异常疼痛产生。
OBJECTIVE: To investigate the role of chloride ion transporters in the production and maintenance of neuropathic pain. Methods: Chronic constriction injury (CCI) was induced in rats. Thermal withdrawal latency (TWL) was measured in rats. Immunohistochemistry and Western Blot were used to detect the expression of sodium-potassium-chloride co-transporter 1 (NKCC1) and potassium-chloride co-transorter2 (KCC2) were expressed on neurons of dorsal root ganglion (DRG) in CCI model The change of chloride ion concentration in DRG neurons was detected by using chloride ion fluorescent probe MQAE (N-Ethoxycarbonylmethyl-6-methoxyquinolinium bromide). Results: (1) The TWL in CCI group was significantly shorter than that in normal control group (P <0.01, n = 10), and still significant at 21 days after operation (P <0.05, n = 10). There was no significant difference between sham operation group and normal group. (2) Under normal circumstances, NKCC1 was weakly expressed in DRG large, medium and small neurons, KCC2 was not expressed. On day 7, day 14 and day 21 after operation, the expression of NKCC1 increased and no expression of KCC2 was observed in CCI model. (3) In the CCI group, the numbers of DRG and NKCC1 positive cells increased significantly (P <0.01, n = 1000) on the 7th postoperative day. , n = 1000). On the 21st postoperative day, the number of midline neurons increased (P <0.01, n = 1000). (4) The expression of NKCC1 protein in DRG neurons increased significantly at 14 and 21 days after CCI. (5) The concentration of chloride ion in DRG neurons was significantly increased at 7 and 14 days after CCI (P <0.05, n = 1000). CONCLUSIONS: In the presence of neuropathic pain, abnormal expression of NKCC1 in DRG neurons increases the intracellular Cl- concentration, which may be involved in the mediation of primary afferent depolarization (PAD) or / and Dorsal root reflexes (DRRs) that induce the distal end of the primary sensory hub trigger hyperalgesia and allodynia.