推拿通过影响NT-3、TrkC表达促进SNI大鼠恢复的机理研究

【作者】 吴剑聪；

【导师】 于天源；

【作者基本信息】 北京中医药大学 ， 针灸推拿学， 2014， 博士

【摘要】 [目的]通过行为学与形态学指标,研究推拿手法促进坐骨神经损伤(sciatic nerve injury,SNI)大鼠神经功能恢复的疗效,再通过神经营养素3(neurotrophin-3, NT-3)、TrkC (tyrosine kinase receptors C,TrkC)在脊髓、背根神经节(dorsal root ganglion,DRG)中的表达情况深入分析推拿修复SNI的起效途径。[方法]以SD大鼠作为实验动物,通过坐骨神经夹持形成大鼠周围神经损伤(peripheral nerve injury,PNI)模型,后用“按摩推拿手法模拟仪”模拟拨法、点法、揉法对推拿组大鼠进行推拿干预,分别在殷门、承山、阳陵泉处治疗,每穴每法各lmin。干预结束后,以光热耐痛阈、坐骨神经功能指数(Sciatic functional index,SFI)、BBB评分评价大鼠感觉、运动功能的恢复情况；再对脊髓、损伤处神经、腓肠肌的形态学切片进行观察分析,寻找推拿手法加强损伤神经再生的证据；最后对脊髓、DRG中NT-3、TrkC的含量进行检测,探讨、阐释推拿促进PNI再生和修复的机理。[结果]1行为学BBB评分结果：造模后7d,模型组大鼠的评分结果显著低于正常组、假手术组,说明造模成功。治疗10、20次后造模3组的评分结果均有所提升,其中推拿组上升最多,显著高于模型组和模型对照组(p<0.05),同时仍低于正常组(p<0.05)。SFI结果：造模后7d,模型组SFI的负值显著低于正常组和假手术组。治疗10次、20次后,模型组、模型对照组和推拿组的负值均显著低于其余2组(p<0.05)。光热耐痛阈结果：在三次行为学检测中,大鼠左足(即正常足)光热耐痛阈结果的组间比较均没有统计学意义。右足的结果则有显著差异。造模后7d,模型组显著高于正常组和假手术组(p<0.05),说明造模导致神经纤维连续性中断,痛觉传导功能受阻。推拿治疗10、20次后,推拿组的抬脚时间明显缩短,低于模型组和模型对照组(p<0.05),同时与正常组相比没有统计学差异(p>0.05),提示推拿可以促进SNI大鼠感觉功能的恢复。2形态学造模后7d,模型组脊髓中存在神经元凋亡,胞体肿胀等,神经则出现变性,可见脱髓鞘、肿胀、破溃等；腓肠肌则见肌细胞直径缩小、肌纤维间隙增宽等失神经支配的表现。治疗10、20次后,造模3组的病变情况均有所缓解,而推拿组修复程度优于模型组和模型对照组,同时推拿组的腓肠肌细胞直径也大于模型组和模型对照组。有髓神经数方面,造模后7d,模型组大鼠患侧有髓神经数显著低于假手术组和模型组(p<0.05)；治疗10次后,造模3组的神经数均有增加,但仍显著低于正常组(p<0.05),同时,模型组、模型对照组均低于推拿组(p<0.05)；治疗20次后的结果与治疗10次后相似,且造模3组的神经数均有增加,其中推拿组更显著。3NT-3、TrkC免疫组化结果在脊髓腹角中,造模后7d,模型组大鼠脊髓前角NT-3的表达已明显高于正常组和假手术组。而推拿组的NT-3水平持续升高,治疗10、20次后均高于其它4组(p<0.05)。模型组和模型对照组则在治疗20次后有所升高,高于正常组(p<0.05)。NT-3在DRG中的表达情况和在脊髓腹角中的表达情况类似。模型组脊髓腹角TrkC造模后7d时低于正常组和假手术组(p<0.05),而在治疗10、20次后造模3组脊髓腹角中TrkC含量均升高,与正常组、假手术组比较均有统计学意义(p<0.05),同时,推拿组的含量高于模型组和模型对照组。在DRG中,造模后7d,模型组的TrkC含量略高于正常组和假手术组(p<0.05),但治疗10、20次后模型组和模型对照组大鼠的TrkC含量均与正常组、假手术组处于相同水平(p>0.05),而推拿组的结果则始终高于其余4组(p<0.05)。[结论]1推拿干预可以促进SNI大鼠的光热耐痛阈、BBB评分、腓肠肌细胞直径的恢复,提示推拿可以改善SNI大鼠的感觉功能和运动功能。2推拿干预可以增加受损神经轴突的有髓神经数,使再生神经与靶器官建立连接,促进功能恢复。3推拿干预可以增加脊髓、DRG中NT-3、TrkC的表达,进而激活NT-3与TrkC结合后的神经元保护和神经再生途径,这可能是推拿促进神经损伤修复的起效机理之一。更多还原

【Abstract】 [Objective]Observe the effect of Tuina therapy to nerve functional recovery of SNI rats through behavioristics, morphology. Then probe the biological mechanisms of Tuina therapy in SNI through the content of NT-3, TrkC in spinal cord and DRG.[Methods]Use SD rats as experimental animals and made the SNI model by nerve clamping method. Rats were divided into5groups. Then rats were treated by manipulation simulator at BL37, BL57and GB34. After treatment, we first tested PWL, SFI, BBB score to evaluated the recovery of sensory and movement function. Then observed morphologic changes in spinal cord, sciatic nerve and gastrocnemius and search the evidence. Finally, detected the content of NT-3, TrkC in spinal cord and DRG to interpreted the mechanism of Tuina to promote nerve regeneration and repair.[Results]1Results of behavioristicsResults of BBB score:7days after operation, the score of the model group were significantly lower than the blank control group and sham-operated group (p <0.05). After10and20treatments, scores of Tuina group, model group and model control group were all higher than before and the score of Tuina group was the highest, which higher than model group and model control group, but still significantly lower than blank control group (p<0.05).Results of SFI:7days after operation, the score of the model group were significantly lower than the blank control group and sham-operated group (p<0.05). After10and20treatments, scores of Tuina group, model group and model control group were still much lower than the other two groups (p>0.05).Results of the PWL test scores:compared the PWL test scores of left foot between each group, no statistical difference. The result of the right foot had statistical difference.7days after operation, the score of the model group was significantly higher than the blank control group and sham-operated group. Indicate that the continuity of nerve fibers had been damaged by operation. The score of Tuina group was significantly lower than model group and model control group after10and20treatments (p<0.05), while no statistical differences compared with normal group (p>0.05). Indicate that Tuina therapy could promote the sensory recovery of SNI rats.2Results of morphologyParts of the neuron in spinal cord of model group dead7days after operation, while some of their cell body swelled etc. Degeneration, demyelination, swell and rupture had been observed in injured nerve. After10and20treatments, pathological changes of model group, model control group and Tuina group were all better than before. The recovery of Tuina group was better. Meanwhile, the diameter of affected gastrocnemius cells of Tuina group was bigger than model group and model control group.Numbers of myelinated nerve fiber of model group was much lower than model group and sham-operated group7days after operation (p<0.05). After10treatments, numbers of myelinated nerve fiber of model group, model control group and Tuina group were higher than before, but still much lower than blank control group (p<0.05). Meanwhile, numbers of model group and model control group were all lower than Tuina group (p<0.05). Result after20treatments is similar to10treatments, and numbers of model group, model control group and Tuina group all increased, numbers of Tuina group increased more.3Results of the NT-3, TrkCIn the spinal cord ventral horn, the NT-3level of model group was higher than blank control group (p<0.05). Meanwhile, the NT-3level of Tuina group continued to rises after10and20treatments and all higher than other four groups (p<0.05). The NT-3levels of model group was increased after20treatments, higher than blank control group but lower than Tuina group (p<0.05).Results of the NT-3in DRG were similar to results in the spinal cord ventral horn.In the spinal cord ventral horn, the TrkC level of model group was higher than blank control group (p<0.05). After10and20treatments, the TrkC levels of model group, model control group and Tuina group were all higher than other two groups (p<0.05). Meanwhile, level of Tuina group was higher than model group and model control group.In DRG, the TrkC level of model group was higher than blank control group7days after operation (p<0.05). After10and20treatments, the TrkC level of Tuina group was higher than other groups (p<0.05).[Conclusion]1Tuina therapy could promote the PWL scores and BBB scores of the SNI rats which indicate that Tuina is effective to improve the sensory and movement function.2Tuina therapy could increase the number of myelinated nerve fiber, rebuild connection between damage nerve axons and the target organ, then promote the recovery of nerve function.3Tuina therapy could increase the level of NT-3and TrkC in spinal cord and DRG, then activated the combination of NT-3and TrkC, finally nerve regeneration started and neurons were protected. This could be a mechanism of Tuina therapy of promoting nerve injury repair.更多还原