推拿对坐骨神经损伤大鼠NGF及其受体选择的研究
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摘要
目的观察推拿对坐骨神经损伤大鼠行为学、形态学、神经生长因子(NGF)、神经营养因子酪氨酸激酶受体A (TrkA)、p75神经营养因子受体(p75NTR)的影响,探讨推拿治疗坐骨神经损伤的起效机理。
方法实验动物选用坐骨神经神经夹持损伤模型大鼠。动物分组采用两次随机分组,分为正常组、假手术组、模型组、模型对照组及推拿组。行为学观察主要采用斜板实验考察感觉功能恢复情况、光热耐痛阈实验考察运动功能恢复情况、体重变化及24h进食量考察大鼠疼痛反应情况；形态学检测主要采用脊髓HE染色及细胞核计数考察神经元恢复情况、腓肠肌HE染色及肌细胞直径测量考察靶组织恢复情况；NGF、TrkA及p75NTR采用免疫组织化学法定性定位分析蛋白表达情况,免疫印迹法进行定量测量蛋白表达研究。免疫组化结果使用Image pro plus6.0图象分析系统,免疫印迹法结果使用Quantity One图像软件分析。所有数据采用SPSS软件统计分析。
结果行为学结果显示：①光热耐痛阈实验,干预0次,造模组与正常组相比明显升高(p<0.05),干预10次及20次后,模型组和模型对照组与正常组相比明显升高(p<0.05),推拿组与模型组相比明显降低(p<0.05),与正常组相比无显著性差异(p>0.05)；②斜板实验,干预0次,造模组与正常组相比明显降低(p<0.05),治疗10次,模型组和模型对照组大鼠斜板实验角度与正常组相比明显降低(p<0.05),推拿组与模型组相比明显升高(p<0.05),与正常组相比有显著性差异(p<0.05),治疗20次,模型组和模型对照组大鼠斜板实验角度与正常组相比明显降低(p<0.05),推拿组与模型组相比明显升高(p<0.05),与正常组相比无显著性差异(p>0.05)；③体重变化：造模后7天(干预0次)后,造模组大鼠体重明显轻于正常组与假手术组(p<0.05),干预10次后,推拿组大鼠体重明显高于模型组和模型对照组(p<0.05),与假手术组无明显差异(p>0.05),但仍低于正常组(p<0.05),干预20次后,推拿组大鼠体重与模型组和模型对照组之间有明显差异(p<0.05),推拿组、假手术组与正常组相比无显著性差异(p>0.05)；④24h进食量：造模后7天(干预0次)后,造模组大鼠进食量明显低于正常组与假手术组(p<0.05)；干预10次后,推拿组大鼠进食量明显高于模型组和模型对照组(p<0.05),与假手术组、正常组无明显差异(p>0.05)；干预20次后,推拿组大鼠进食量与模型组和模型对照组之间有明显差异(p<0.05),推拿组、正常组之间相比无显著性差异(p>0.05),推拿组大鼠进食量高于假手术组(p<0.05)。
形态学观察：①脊髓HE染色细胞核计数比较,造模后7天(干预0次)后,造模组明显低于正常组与假手术组(p<0.05),正常组与假手术组间无明显差异(p>0.05),干预10次后,推拿组大鼠明显高于模型组和模型对照组(p<0.05),低于假手术组和正常组(p<0.05),模型组、模型对照组与正常组相比也有统计学意义(p<0.05),干预20次后,推拿组大鼠与模型组和模型对照组之间有明显差异(p<0.05)；模型组和模型对照组明显低于正常组(p<0.05)；推拿组、正常组之间相比无显著性差异(p>0.05)。②腓肠肌HE染色肌细胞直径比较：造模后7天(干预0次),造模组大鼠肌细胞直径明显低于低于假手术组和正常组(p<0.05)；正常组与假手术组间无明显差异(p>0.05),干预10次后,推拿组大鼠与模型组和模型对照组之间有明显差异(p<0.05)；模型组和模型对照组明显低于正常组(p<0.05)；推拿组仍低于正常组(p<0.05),干预20次后,推拿组与其余四组均有显著差异(p<0.05),高于模型组和模型对照组(p<0.05),与正常组无显著差异(p>0.05)；模型组和模型对照组明显低于正常组(p<0.05)。
NGF免疫组化及wB结果显示：干预0次,模型组与正常组较正常组明显升高(p<0.05),干预10次及20次后,模型组和模型对照组NGF积分光密度与正常组相比明显升高(p<0.05),推拿组与模型组相比明显升高(p<0.05),与正常组相比明显升高(p<0.05)。
TrkA免疫组化及wB结果显示：干预0次,模型组与正常组较正常组明显升高(p<0.05),干预10次及20次后,模型组和模型对照组TrkA积分光密度与正常组相比明显升高(p<0.05),推拿组与模型组相比明显升高(p<0.05),与正常组相比明显升高(p<0.05)。
p75NTR免疫组化及wB结果显示：干预0次,模型组与正常组较正常组明显升高(p<0.05),干预10次及20次后,模型组和模型对照组p75NTR积分光密度与正常组相比明显升高(p<0.05),推拿组与模型组相比明显升高(p<0.05),与正常组相比明显升高(p<0.05)。
结论推拿治疗可以有效的促进坐骨神经损伤大鼠的感觉及运动功能,对大鼠的疼痛反应也具有良好的恢复效果,可促进坐骨神经损伤大鼠脊髓中NGF表达,并提高NGF高亲和受体TrkA的表达,降低低亲和力受体p75NTR的表达,从而激活NGF与TrkA结合后介导的神经再生机制,抑制NGF与p75NTR结合后介导的神经凋亡机制。推拿治疗影响NGF及其受体的表达含量,进而影响NCF对受体的选择,是推拿治疗周围神经损伤的起效机理之一
Objective
Observe the influence of Tuina therapy to the behavioristics, morphology, NGF, TrkA and p75NTR of sciatic nerve injury model rats. Probe the biological mechanisms of Tuina therapy in sciatic nerve injury.
Method
Use the nerve clamping method to make the sciatic nerve injury model. All the rats are devided into5groups with two-time randomgroup design.5groups are blank control group,-sham-operated group, model control group, sham-Tuina group and Tuina group. Behavioral indexes including PWL test, board test, weight, food consumption in24h. Morphological indexes including HE of medulla spinalis and gastrocnemius muscle, counting of medulla spinalis nucleus and diameter of gastrocnemius muscle cells. NGF, TrkA and p75NTR is measured by immunohistochemical method as result of qualitative analyses. NGF, TrkA and p75NTR is measured by western blot method as result of quantitative analyses. Immunohistochemical results are analysed by Image pro plus6.0. Western blot results are analysed by Quantity One. All the datas are analysed by SPSS.
Result
The PWL test scores of the model group are higher than the blank control group in0,10and20times of treatments (P<0.05). Tuina group's score is much lower in10and20times compared with model group (P<0.05). The board test scores of the model group are lower than the blank control group in0,10and20times of treatments (P<0.05). Tuina group's score is much higher in10and20times compared with model group(P<0.05). The weights of model group rats are lower than the blank control group in0,10and20times (P<0.05). Tuina group rats' weights are higher than model group (P<0.05). Food consumption result in24h shows the same ones of weights. In counting results of medulla spinalis nucleus, Tuina group is higher than model group in10and20times (P<0.05). In diameter results of gastrocnemius muscle cells, Tuina group is lower than model group in10and20times (P<0.05). In the result of NGF and TrkA, the model group is higher than blank control group(P<0.05), tuina group is higher than model group (P<0.05). In the result of p75NTR, the model group is higher than blank control group (P<0.05), tuina group is lower than blank control group (P<0.05).
Conclusion
Tuina treatment is effective to improve the feeling function and motor function of the sciatic nerve injury rats. Tuina is effective to reduce the pain of rats. NGF, TrkA of medulla spinalis are improved by tuina. p75NTR of medulla spinalis is reduced by tuina. In this case, NGF and TrkA would start the nerve regenerat ion mechanism. In the other hand, NGF and p75NTR would stop the the neural apoptosis mechanism. As a result, tuina can change the NGF and its receptor. NGF would choose different receptor by tuina treatment. This could be one of the mechanisms of tuina's effects.
方法实验动物选用坐骨神经神经夹持损伤模型大鼠。动物分组采用两次随机分组,分为正常组、假手术组、模型组、模型对照组及推拿组。行为学观察主要采用斜板实验考察感觉功能恢复情况、光热耐痛阈实验考察运动功能恢复情况、体重变化及24h进食量考察大鼠疼痛反应情况；形态学检测主要采用脊髓HE染色及细胞核计数考察神经元恢复情况、腓肠肌HE染色及肌细胞直径测量考察靶组织恢复情况；NGF、TrkA及p75NTR采用免疫组织化学法定性定位分析蛋白表达情况,免疫印迹法进行定量测量蛋白表达研究。免疫组化结果使用Image pro plus6.0图象分析系统,免疫印迹法结果使用Quantity One图像软件分析。所有数据采用SPSS软件统计分析。
结果行为学结果显示：①光热耐痛阈实验,干预0次,造模组与正常组相比明显升高(p<0.05),干预10次及20次后,模型组和模型对照组与正常组相比明显升高(p<0.05),推拿组与模型组相比明显降低(p<0.05),与正常组相比无显著性差异(p>0.05)；②斜板实验,干预0次,造模组与正常组相比明显降低(p<0.05),治疗10次,模型组和模型对照组大鼠斜板实验角度与正常组相比明显降低(p<0.05),推拿组与模型组相比明显升高(p<0.05),与正常组相比有显著性差异(p<0.05),治疗20次,模型组和模型对照组大鼠斜板实验角度与正常组相比明显降低(p<0.05),推拿组与模型组相比明显升高(p<0.05),与正常组相比无显著性差异(p>0.05)；③体重变化：造模后7天(干预0次)后,造模组大鼠体重明显轻于正常组与假手术组(p<0.05),干预10次后,推拿组大鼠体重明显高于模型组和模型对照组(p<0.05),与假手术组无明显差异(p>0.05),但仍低于正常组(p<0.05),干预20次后,推拿组大鼠体重与模型组和模型对照组之间有明显差异(p<0.05),推拿组、假手术组与正常组相比无显著性差异(p>0.05)；④24h进食量：造模后7天(干预0次)后,造模组大鼠进食量明显低于正常组与假手术组(p<0.05)；干预10次后,推拿组大鼠进食量明显高于模型组和模型对照组(p<0.05),与假手术组、正常组无明显差异(p>0.05)；干预20次后,推拿组大鼠进食量与模型组和模型对照组之间有明显差异(p<0.05),推拿组、正常组之间相比无显著性差异(p>0.05),推拿组大鼠进食量高于假手术组(p<0.05)。
形态学观察：①脊髓HE染色细胞核计数比较,造模后7天(干预0次)后,造模组明显低于正常组与假手术组(p<0.05),正常组与假手术组间无明显差异(p>0.05),干预10次后,推拿组大鼠明显高于模型组和模型对照组(p<0.05),低于假手术组和正常组(p<0.05),模型组、模型对照组与正常组相比也有统计学意义(p<0.05),干预20次后,推拿组大鼠与模型组和模型对照组之间有明显差异(p<0.05)；模型组和模型对照组明显低于正常组(p<0.05)；推拿组、正常组之间相比无显著性差异(p>0.05)。②腓肠肌HE染色肌细胞直径比较：造模后7天(干预0次),造模组大鼠肌细胞直径明显低于低于假手术组和正常组(p<0.05)；正常组与假手术组间无明显差异(p>0.05),干预10次后,推拿组大鼠与模型组和模型对照组之间有明显差异(p<0.05)；模型组和模型对照组明显低于正常组(p<0.05)；推拿组仍低于正常组(p<0.05),干预20次后,推拿组与其余四组均有显著差异(p<0.05),高于模型组和模型对照组(p<0.05),与正常组无显著差异(p>0.05)；模型组和模型对照组明显低于正常组(p<0.05)。
NGF免疫组化及wB结果显示：干预0次,模型组与正常组较正常组明显升高(p<0.05),干预10次及20次后,模型组和模型对照组NGF积分光密度与正常组相比明显升高(p<0.05),推拿组与模型组相比明显升高(p<0.05),与正常组相比明显升高(p<0.05)。
TrkA免疫组化及wB结果显示：干预0次,模型组与正常组较正常组明显升高(p<0.05),干预10次及20次后,模型组和模型对照组TrkA积分光密度与正常组相比明显升高(p<0.05),推拿组与模型组相比明显升高(p<0.05),与正常组相比明显升高(p<0.05)。
p75NTR免疫组化及wB结果显示：干预0次,模型组与正常组较正常组明显升高(p<0.05),干预10次及20次后,模型组和模型对照组p75NTR积分光密度与正常组相比明显升高(p<0.05),推拿组与模型组相比明显升高(p<0.05),与正常组相比明显升高(p<0.05)。
结论推拿治疗可以有效的促进坐骨神经损伤大鼠的感觉及运动功能,对大鼠的疼痛反应也具有良好的恢复效果,可促进坐骨神经损伤大鼠脊髓中NGF表达,并提高NGF高亲和受体TrkA的表达,降低低亲和力受体p75NTR的表达,从而激活NGF与TrkA结合后介导的神经再生机制,抑制NGF与p75NTR结合后介导的神经凋亡机制。推拿治疗影响NGF及其受体的表达含量,进而影响NCF对受体的选择,是推拿治疗周围神经损伤的起效机理之一
Objective
Observe the influence of Tuina therapy to the behavioristics, morphology, NGF, TrkA and p75NTR of sciatic nerve injury model rats. Probe the biological mechanisms of Tuina therapy in sciatic nerve injury.
Method
Use the nerve clamping method to make the sciatic nerve injury model. All the rats are devided into5groups with two-time randomgroup design.5groups are blank control group,-sham-operated group, model control group, sham-Tuina group and Tuina group. Behavioral indexes including PWL test, board test, weight, food consumption in24h. Morphological indexes including HE of medulla spinalis and gastrocnemius muscle, counting of medulla spinalis nucleus and diameter of gastrocnemius muscle cells. NGF, TrkA and p75NTR is measured by immunohistochemical method as result of qualitative analyses. NGF, TrkA and p75NTR is measured by western blot method as result of quantitative analyses. Immunohistochemical results are analysed by Image pro plus6.0. Western blot results are analysed by Quantity One. All the datas are analysed by SPSS.
Result
The PWL test scores of the model group are higher than the blank control group in0,10and20times of treatments (P<0.05). Tuina group's score is much lower in10and20times compared with model group (P<0.05). The board test scores of the model group are lower than the blank control group in0,10and20times of treatments (P<0.05). Tuina group's score is much higher in10and20times compared with model group(P<0.05). The weights of model group rats are lower than the blank control group in0,10and20times (P<0.05). Tuina group rats' weights are higher than model group (P<0.05). Food consumption result in24h shows the same ones of weights. In counting results of medulla spinalis nucleus, Tuina group is higher than model group in10and20times (P<0.05). In diameter results of gastrocnemius muscle cells, Tuina group is lower than model group in10and20times (P<0.05). In the result of NGF and TrkA, the model group is higher than blank control group(P<0.05), tuina group is higher than model group (P<0.05). In the result of p75NTR, the model group is higher than blank control group (P<0.05), tuina group is lower than blank control group (P<0.05).
Conclusion
Tuina treatment is effective to improve the feeling function and motor function of the sciatic nerve injury rats. Tuina is effective to reduce the pain of rats. NGF, TrkA of medulla spinalis are improved by tuina. p75NTR of medulla spinalis is reduced by tuina. In this case, NGF and TrkA would start the nerve regenerat ion mechanism. In the other hand, NGF and p75NTR would stop the the neural apoptosis mechanism. As a result, tuina can change the NGF and its receptor. NGF would choose different receptor by tuina treatment. This could be one of the mechanisms of tuina's effects.
引文
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