推拿对坐骨神经损伤大鼠生长相关蛋白表达影响的研究

【作者】 周嫱；

【导师】 于天源；

【作者基本信息】 北京中医药大学 ， 针灸推拿学， 2012， 硕士

【摘要】 [目的]基于推拿治疗坐骨神经损伤具有良好的临床疗效,从实验角度探寻坐骨神经损伤经推拿治疗后,机体在感觉功能及运动功能方面的改善效应,并通过对背根神经节中生长相关蛋白表达变化的研究,进一步揭示推拿治疗坐骨神经损伤的起效机理及作用途径。[方法]以SD大鼠为实验动物,制备坐骨神经夹持损伤模型,选取患侧殷门穴、承山穴、阳陵泉穴,每天用按摩手法模拟仪于以上三穴施以揉法、点法和拨法,通过对其斜板试验和足底部光热耐痛阈的检测,从行为学角度探寻坐骨神经损伤经推拿治疗后肢体运动功能和感觉功能的恢复情况；通过观察背根神经节脊髓、坐骨神经以及腓肠肌HE染色的变化,从形态学角度探寻推拿对神经元再生修复的促进效应；通过检测背根神经节及腓肠肌中生长相关蛋白表达的变化,探寻推拿促进损伤神经再生修复的机制,揭示推拿治疗坐骨神经损伤的起效机理和作用途径。[结果]1.行为学造模7天后,模型组大鼠的斜板试验结果下降,与正常组比较具有统计学意义。推拿治疗10天后,推拿治疗组斜板试验结果高于模型组、模型对照组,趋于正常。推拿治疗可以使坐骨神经损伤大鼠腓肠肌肌力明显改善,促进其运动功能的恢复。造模7天后,模型组大鼠右侧足底部光热耐痛阈升高,与正常组比较有统计学意义。推拿治疗10天后,推拿治疗组大鼠右侧足底部光热耐痛阈明显低于模型组,高于正常组,与模型组有统计学意义。推拿治疗可以提高坐骨神经损伤大鼠的痛觉敏感程度,促进其感觉功能的恢复。2.病理学正常情况下,背根神经节HE染色可见其神经元多数呈圆形,胞质呈嗜酸性,体积较大；胞核呈嗜碱性,染色浅,大而圆；核仁清楚,胞体外周有卫星细胞包绕；脊髓HE染色可见脊髓腹角细胞胞核位于细胞中央,尼氏体均匀分布。通过对模型组的观察,DRG内部分神经元出现中央染色质溶解的现象,神经元有不同程度的坏死,细胞出现肿胀、变形和溶解的现象,胞浆的染色不均匀,细胞的形态也大小不一；脊髓HE染色显示细胞出现肿胀、胞核移向边缘尼氏体溶解消失等变性现象。说明坐骨神经损伤后,会导致背根神经节和脊髓腹角运动神经元胞体死亡。通过对推拿治疗组的观察,DRG内神经元形状渐规则,大多为圆形,细胞肿胀及变形的现象不明显；脊髓腹角神经元部分细胞核移向边缘,部分细胞核位于中央,尼氏体溶解,细胞排列在完整性、方向性等方面好于模型组和模型对照组。说明推拿对坐骨神经损伤后感觉神经元和运动神经元的修复具有一定的促进作用。3.生长相关蛋白正常组及假手术组大鼠GAP-43在背根神经节及腓肠肌中有少量的表达,阳性的胞浆呈浅棕褐色,胞核呈淡蓝色；神经损伤7天后,模型组及模型对照组大鼠GAP-43在背根神经节及腓肠肌中的表达情况较正常组染色加深,免疫组化平均光密度增高,存在差异；推拿治疗10天后,模型组及模型对照组大鼠GAP-43在背根神经节及腓肠肌中的免疫组化平均光密度较正常组有一定程度增高,推拿治疗组大鼠GAP-43在背根神经节及腓肠肌中免疫组化平均光密度显著增高,染色程度和范围明显优于模型组,表达情况更为明显,较其余四组均存在差异。说明推拿治疗可以促进GAP-43在背根神经节及腓肠肌中的表达。[结论]推拿可以提高坐骨神经损伤大鼠的痛觉敏感程度,提高患侧腓肠肌的肌力及肌肉恢复率,从而促进坐骨神经损伤大鼠运动功能和感觉功能的恢复；其作用途径为对背根神经节感觉神经元及脊髓腹角运动神经元的营养和保护；起效机理之一为促进背根神经节及腓肠肌中生长相关蛋白的表达以促进损伤神经的再生修复,为损伤神经提供良好的微环境。更多还原

【Abstract】 【Objective】Based on better clinical effect of massage therapy on sciatic nerve injury, this study is to research the recovery on both sensory and motor function after massage therapy. Meanwhile, through the research on changes of GAP-43expression in DRG, the study can explore massage’s approach and central mechanism on the treatment of sciatic nerve injury.【Methods】Taking SD rats as experimental animals to build clamping injured model, the study used massage simulator on UB37, BL57and GB34. And also, solar-thermal pain threshold resistance and inclined plate test is used from a behavioral point of view to explore the recovery on sensory and motor function after therapy. Through observing the variation of DRG, the spinal marrow, sciatic nerve and the HE of gastrocnemius, the promotion of therapy’s effect on neuronal regeneration is sought on aspect of morphology. Through the research on changes of GAP-43expression in DRG and gastrocnemius muscles, approach and mechanism is revealed by massage therapy on sciatic nerve injury.【Results】1. EthologySeven days after made the model, results of inclined plate group declined which has statistical significance compared with normal group. Ten days after massage therapy, curative effect of inclined plate group is better than sham operation group, model group and control group. It means that massage therapy can improve gastrocnemius muscle for the sciatic nerve injury and promote the recovery of motor function.Seven days after made the model, solar-thermal pain threshold resistance in model group is increased and it has statistical significance compared with normal group. Ten days after massage therapy, solar-thermal pain threshold resistance in therapy group is obviously lower than model group and higher than normal group. It has statistical significance compared with model group. It showed massage therapy can improve pain sensitivity for the sciatic nerve injury and promote the recovery of sensory function.2. PathologyUnder normal circumstances, the neuron of DRG HE stain is visibly round and cytoplasm is eosinophilic and the cell volume is a bit larger. Cell nucleus is basophilic, staining light, large and round. Cell nucleolus is clear with satellite cells enveloping. HE in the spinal marrow is visible and cell nucleus is located in the central cells. Nissl body distributed uniformly of the cell bodies.After sciatic nerve injured, part of DRG neurons appeared that chromatin dissolved. Neurons have different degrees of necrosis. Some cells were swelled and dissolved with some deformation. Cytoplasm staining wasn’t even with form size differ. HE staining of the spinal cord cell swelled with degeneration phenomenon. The nucleus moved toward the edge and nissl body dissolved and disappeared. It showed that after sciatic nerve has injured, DRG and motor neuron cell of spinal cord ventral-horn died.After massage therapy, DRG neuron is in the shape of gradual rules, mostly round. Cell is swelled and deformation phenomenon is not obvious. Part of ventral-horn nerve nucleus moved toward the edge and part of the nucleus is located in the central. Nissl body dissolved, cells array is better than model group and model control group in integrity and direction. Massage therapy can promote the recovery of both sensory and motor neuron after the sciatic nerve injured.3. Growth associated proteinGAP-43in normal group and sham operation group has some expression in DRG and gastrocnemius muscles. Positive neurons cytoplasm appeared light brown, nucleus appeared pale blue. After seven days of sciatic nerve injured, GAP-43expression from model group and model control group in DRG and gastrocnemius muscles is deepen staining than normal group. AOD was increased, where there are differences. After ten days of massage therapy, AOD of GAP-43from model group and model control group in DRG and gastrocnemius muscles has a bit increase than normal group. AOD of GAP-43in DRG and AOD of GAP-43in gastrocnemius from massage therapy group significantly increased, staining extent and scope significantly better than model group. The expression is more obvious than the other four groups which has statistical significance.[Conclusion]Massage therapy can promote pain sensitivity of sciatic nerve injured rats, it also can increase gastrocnemius muscle’s recovery rate. Both sensory and motor function is enhanced on the sciatic nerve injury. It effected on sensory neurons in DRG and motor neurons in the spinal marrow. The mechanism is for the promotion of GAP-43expression in both DRG and gastrocnemius muscles’growth-related proteins to promote the regeneration of injured nerves, and provide a good microcnvironment for the injured nerve.更多还原