GDF10在神经病理性疼痛大鼠脊髓中的表达变化及机制研究
发布时间：2019-03-17 09:26
【摘要】：由中枢和周围神经系统损伤导致的神经病理性疼痛(neuropathic pain,NP)是一种难治性疾病,目前的治疗方法大都以暂时性的止痛和理疗为主,但并没有实现长期的治疗疼痛的目的,加强神经病理性疼痛发生机制的研究对其开发新的治疗药物及方法有重要意义。目前关于神经病理性疼痛的发生机制主要有神经炎症反应以及中枢敏化。转化生长因子β(transforming growth factorβ,TGF-β)超家族是一类具有多功能并且具有环境依赖性的细胞因子,其亚家族包括TGF-βs、骨形态发生蛋白(bone morphogenetic proteins,BMPs)、活化素(activins)、生长和分化因子(growth and differentiation factors,GDFs)等,其中许多成员(如TGF-β1)具有神经炎症抑制作用,可以减轻神经病理性疼痛的发生。生长和分化因子10(growth and differentiation factor 10,GDF10)是BMPs亚家族成员之一,它在神经发生以及再生中起着重要作用,通过在GEO数据库中查询显示神经病理性疼痛大鼠的背根神经节中GDF10的表达增加,而有关其在神经病理性疼痛发生时在脊髓中的变化及作用尚未见报道。N-甲基-D-天冬氨酸(N-methyl-D-aspartate,NMDA)受体的活化而引起的脊髓中枢敏化在神经病理性疼痛的发生中有着重要作用,由此我们进一步研究GDF10是否与NMDA受体的活化间有关联,从而了解GDF10在神经病理性疼痛中的相关机制。方法:(1)大鼠神经病理性疼痛模型的建立。采用结扎雄性SD大鼠左侧L5脊神经的方法建立此模型。(2)药物鞘内注射。采用鞘内注射的方法对大鼠注射NMDA使其产生神经病理性疼痛,并对脊神经结扎大鼠鞘内注射NMDA受体的拮抗剂MK-801。(3)行为学检测。用von Frey filaments对大鼠缩爪阈值进行测量,以反应后肢足底的机械痛敏的变化。(4)Western Blot。利用免疫印迹检测脊神经结扎大鼠术侧脊髓在术后1d、3d、10d、21d GDF10蛋白表达的变化,并检测NMDA鞘内注射后脊髓内GDF10的表达变化以及鞘内注射MK-801对脊神经结扎大鼠脊髓背角GDF10的表达。(4)免疫荧光。用免疫荧光检测脊神经结扎后脊髓背角中GDF10在术后3d、10d、21d的表达变化及NMDA鞘内注射后GDF10的表达,并通过免疫荧光双标检测GDF10在脊髓中的细胞定位。主要结果:1.脊神经结扎大鼠在术后第1 d缩爪阈值开始降低,3 d后与正常对照组相比,各时相点差异比较均有统计学意义(P0.05),到第10 d阈值下降最明显,至21 d呈现持平状态,大鼠呈现出明显的机械痛敏增加,表明成功建立了神经病理性疼痛大鼠模型。2.免疫荧光双标检测观察到正常大鼠L5脊髓组织中GDF10与神经元标记物NeuN共表达,而不与星形胶质细胞标记物GFAP或小胶质细胞标记物Iba-1共表达。Western blot证实脊神经结扎后术侧脊髓中GDF10蛋白的表达逐渐降低,至第10 d减少最为显著,至21d呈现低水平表达状态,与假手术组相比差异具有统计学意义(P0.05)。免疫荧光显示术侧脊髓背角神经元内GDF10表达在3d、10d、21d逐渐降低,与western blot结果相一致。3.正常大鼠鞘内注射NMDA,在连续3d的注射中,同一时间点与鞘内注射生理盐水组大鼠相比,大鼠缩爪阈值显著降低(P0.05);western blot检测显示NMDA组大鼠脊髓背角内GDF10表达较生理盐水组显著降低(P0.05);免疫荧光检测发现NMDA鞘内注射能显著减少脊髓背角神经元内GDF10的表达。4.脊神经结扎大鼠鞘内注射MK-801,能显著抑制脊神经结扎引起的缩爪阈值的降低,与脊神经结扎大鼠鞘内注射生理盐水组相比有统计学意义(P0.05)。western blot检测发现脊神经结扎大鼠鞘内注射MK-801能显著抑制脊髓内GDF10的表达减少,与生理盐水组相比有统计学意义(P0.05)。结论:(1)采用左侧L5脊神经结扎成功建立了大鼠神经病理性疼痛模型;(2)脊神经结扎使大鼠机械痛敏增加,并使脊髓背角神经元内GDF10的表达减少;(3)鞘内注射NMDA使大鼠机械痛敏显著增加,并使大鼠脊髓背角神经元内GDF10表达减少,表明脊髓内NMDA受体的活化与GDF10的表达减少相关;(4)鞘内注射MK-801抑制脊神经结扎大鼠脊髓内NMDA受体的活化可以减轻神经病理性疼痛,并可抑制脊髓内GDF10的表达减少。
[Abstract]:Neuropathic pain (NP), which is caused by the damage of the central and peripheral nervous system, is a refractory disease. The study of strengthening the pathogenesis of neuropathic pain is of great significance to the development of new therapeutic drugs and methods. At present, the mechanism of the occurrence of neuropathic pain is mainly the reaction of the nerve inflammation and the central sensitization. Transformed growth factor (TGF-1) superfamily is a class of cytokines with multi-function and environment-dependent, the subfamily of which includes TGF-Fass, bone morphogenic proteins (BMPs), activins, growth and differentiation factors. GDFs et al., many of which, such as TGF-(1), have a neuroinflammatory inhibitory effect and can reduce the occurrence of neuropathic pain. Growth and differentiation factor 10 (GDF10) is one of the members of the BMPs subfamily, which plays an important role in the genesis and regeneration of the nerve. The changes of the spinal cord in the spinal cord and its role in the occurrence of neuropathic pain have not been reported. The activation of N-methyl-D-aspartate (NMDA) receptor plays an important role in the pathogenesis of neuropathic pain, and we further study whether the GDF10 is associated with the activation of the NMDA receptor, So as to understand the relevant mechanism of the GDF10 in the neuropathic pain. Methods: (1) The model of neuropathic pain in rats was established. This model was established using a method of ligation of the left L5 spinal nerve in male SD rats. (2) Intra-drug injection. The rats were injected with NMDA to produce neuropathic pain and the antagonist MK-801 of the NMDA receptor was injected into the rat with spinal nerve ligation. (3) Behavior detection. The rat paw threshold was measured with von Frey film to respond to changes in mechanical pain in the foot of the hindlimb. (4)Western Blot. The expression of GDF10 and the expression of GDF10 in the spinal cord of rats were detected by immunoblotting. The expression of GDF10 in the spinal cord and the expression of the dorsal horn GDF10 of the spinal cord in the spinal cord of the rats were detected by the injection of MK-801 in the spinal cord. (4) immunofluorescence. The expression of GDF10 and the expression of GDF10 in the spinal dorsal horn of spinal cord after spinal nerve ligation were detected by immunofluorescence, and the cellular localization of the GDF10 in the spinal cord was detected by immunofluorescence double-label. Main results:1. The threshold of the first day after the operation of the spinal nerve was decreased, and the difference of the point-to-point difference was statistically significant after 3 days (P <0.05). The decrease of the threshold of the 10th day was the most obvious, and the level of 21 d was in the same state, and the rats exhibited a significant increase in the mechanical pain. It was shown that the model of neuropathic pain was successfully established. The co-expression of the GDF10 with the neuronal marker NeuN was observed in the normal rat L5 spinal cord tissue without co-expression with the astrocyte marker GFAP or the microglial cell marker Iba-1. Western blot confirmed that the expression of the GDF10 protein in the spinal cord after the spinal nerve ligation was gradually decreased, the most significant in the 10th day, and the expression of the low-level expression in the 21 d was statistically significant compared with the sham-operated group (P0.05). The results showed that the expression of GDF10 in the dorsal horn of the spinal dorsal horn of the lateral spinal cord decreased gradually in the 3-d,10-day, and 21-day, and was consistent with the western blot. In the normal rat, NMDA was injected into the rat, and the threshold of the rat's paw was significantly lower than that of the normal saline group (P <0.05), and the expression of GDF10 in the dorsal horn of the spinal cord of the NMDA group was significantly lower in the spinal dorsal horn of the rats (P0.05). The expression of GDF10 in the dorsal horn neurons of the spinal cord was significantly reduced by immunofluorescent assay. The MK-801 was injected into the rat with spinal nerve ligation, which can significantly inhibit the reduction of the threshold of the contraction claw caused by the ligature of the spinal nerve. Compared with the saline group, the expression of MK-801 in the rat spinal cord was significantly inhibited by Western blot, and the expression of GDF10 in the spinal cord was significantly inhibited. Conclusion: (1) The rat's neuropathic pain model was successfully established with the left L5 spinal nerve ligation; (2) the spinal nerve ligation increased the mechanical pain of the rat and decreased the expression of the GDF10 in the dorsal horn of the spinal cord; and (3) the intraventricular injection of NMDA increased the mechanical pain of the rat. and the expression of the GDF10 in the dorsal horn of the spinal cord of the rat is reduced, the activation of the NMDA receptor in the spinal cord is related to the reduction of the expression of the GDF10, and (4) the activation of the NMDA receptor in the spinal cord of the rat spinal cord is inhibited by the intra-spinal injection of the MK-801, and the neuropathic pain can be reduced, And the expression of the GDF10 in the spinal cord can be reduced.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R402
本文编号：2442154
[Abstract]:Neuropathic pain (NP), which is caused by the damage of the central and peripheral nervous system, is a refractory disease. The study of strengthening the pathogenesis of neuropathic pain is of great significance to the development of new therapeutic drugs and methods. At present, the mechanism of the occurrence of neuropathic pain is mainly the reaction of the nerve inflammation and the central sensitization. Transformed growth factor (TGF-1) superfamily is a class of cytokines with multi-function and environment-dependent, the subfamily of which includes TGF-Fass, bone morphogenic proteins (BMPs), activins, growth and differentiation factors. GDFs et al., many of which, such as TGF-(1), have a neuroinflammatory inhibitory effect and can reduce the occurrence of neuropathic pain. Growth and differentiation factor 10 (GDF10) is one of the members of the BMPs subfamily, which plays an important role in the genesis and regeneration of the nerve. The changes of the spinal cord in the spinal cord and its role in the occurrence of neuropathic pain have not been reported. The activation of N-methyl-D-aspartate (NMDA) receptor plays an important role in the pathogenesis of neuropathic pain, and we further study whether the GDF10 is associated with the activation of the NMDA receptor, So as to understand the relevant mechanism of the GDF10 in the neuropathic pain. Methods: (1) The model of neuropathic pain in rats was established. This model was established using a method of ligation of the left L5 spinal nerve in male SD rats. (2) Intra-drug injection. The rats were injected with NMDA to produce neuropathic pain and the antagonist MK-801 of the NMDA receptor was injected into the rat with spinal nerve ligation. (3) Behavior detection. The rat paw threshold was measured with von Frey film to respond to changes in mechanical pain in the foot of the hindlimb. (4)Western Blot. The expression of GDF10 and the expression of GDF10 in the spinal cord of rats were detected by immunoblotting. The expression of GDF10 in the spinal cord and the expression of the dorsal horn GDF10 of the spinal cord in the spinal cord of the rats were detected by the injection of MK-801 in the spinal cord. (4) immunofluorescence. The expression of GDF10 and the expression of GDF10 in the spinal dorsal horn of spinal cord after spinal nerve ligation were detected by immunofluorescence, and the cellular localization of the GDF10 in the spinal cord was detected by immunofluorescence double-label. Main results:1. The threshold of the first day after the operation of the spinal nerve was decreased, and the difference of the point-to-point difference was statistically significant after 3 days (P <0.05). The decrease of the threshold of the 10th day was the most obvious, and the level of 21 d was in the same state, and the rats exhibited a significant increase in the mechanical pain. It was shown that the model of neuropathic pain was successfully established. The co-expression of the GDF10 with the neuronal marker NeuN was observed in the normal rat L5 spinal cord tissue without co-expression with the astrocyte marker GFAP or the microglial cell marker Iba-1. Western blot confirmed that the expression of the GDF10 protein in the spinal cord after the spinal nerve ligation was gradually decreased, the most significant in the 10th day, and the expression of the low-level expression in the 21 d was statistically significant compared with the sham-operated group (P0.05). The results showed that the expression of GDF10 in the dorsal horn of the spinal dorsal horn of the lateral spinal cord decreased gradually in the 3-d,10-day, and 21-day, and was consistent with the western blot. In the normal rat, NMDA was injected into the rat, and the threshold of the rat's paw was significantly lower than that of the normal saline group (P <0.05), and the expression of GDF10 in the dorsal horn of the spinal cord of the NMDA group was significantly lower in the spinal dorsal horn of the rats (P0.05). The expression of GDF10 in the dorsal horn neurons of the spinal cord was significantly reduced by immunofluorescent assay. The MK-801 was injected into the rat with spinal nerve ligation, which can significantly inhibit the reduction of the threshold of the contraction claw caused by the ligature of the spinal nerve. Compared with the saline group, the expression of MK-801 in the rat spinal cord was significantly inhibited by Western blot, and the expression of GDF10 in the spinal cord was significantly inhibited. Conclusion: (1) The rat's neuropathic pain model was successfully established with the left L5 spinal nerve ligation; (2) the spinal nerve ligation increased the mechanical pain of the rat and decreased the expression of the GDF10 in the dorsal horn of the spinal cord; and (3) the intraventricular injection of NMDA increased the mechanical pain of the rat. and the expression of the GDF10 in the dorsal horn of the spinal cord of the rat is reduced, the activation of the NMDA receptor in the spinal cord is related to the reduction of the expression of the GDF10, and (4) the activation of the NMDA receptor in the spinal cord of the rat spinal cord is inhibited by the intra-spinal injection of the MK-801, and the neuropathic pain can be reduced, And the expression of the GDF10 in the spinal cord can be reduced.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R402
【参考文献】
相关期刊论文 前1条
1 刘国凯,黄宇光,罗爱伦;神经病理性疼痛动物模型及其评价[J];中国临床药理学与治疗学;2005年06期
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