“温阳补气”针法对EAMG大鼠RNS衰减值及血清AchR表达水平影响的研究
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摘要
目的:通过AchR片段129～145建立重症肌无力经典动物模型—实验性自身免疫性重症肌无力(Experimental autoimmune myasthenia gravis-EAMG)大鼠,通过“温阳补气”针法对EAMG大鼠低频重复神经电刺激(repetitive nerve stimulation,RNS)及血清乙酰胆碱受体(Acetylcholine receptors AchR)影响的研究,探讨“温阳补气”针法对EAMG大鼠治疗作用及作用机制。进一步丰富了中医治痿理论,为自身免疫性疾病的治疗及机制的探讨提供了新的手段。
方法:实验选取Lewis大鼠,将AchR片段129～145以1:2的比例与完全弗氏佐剂(CFA)完全乳化,建立EAMG大鼠模型。上述抗原乳剂以200μl/只注射于大鼠四足垫部(50μl),抗原量为50μg/只。分别于初次免疫后第4周、第8周,再次同法免疫接种。记录大鼠的体重变化,参考Lennon的评分标准评价模型症状等级,RNS衰减值和血清AchR-ab表达水平判定模型是否成功。选取造模成功大鼠30只,随机分为模型组、溴吡斯的明组、“温阳补气”针法组。空白组同期同条件饲养,相同条件下抓捕与处理;模型组大鼠也是同期同条件饲养,不予任何治疗;“温阳补气”针法组给予“温阳补气”针法组治疗;溴吡斯的明组给予溴吡斯的明治疗。分别于发病后的第1天及干预后第14天应用Oxford-肌电图诱发电位仪检测RNS衰减值变化;应用酶联免疫吸附法(ELISA法)检测AchR表达水平变化,并每周观察EAMG大鼠症状和体重变化。
结果:1.应用70只大鼠建立模型,成功32只(成功后死亡2只),成功率为45.71%。2.EAMG大鼠体重增加缓慢,表现出较为明显的肌无力症状,临床症状评分为2.150,与空白组比较有明显性差异(p<0.05)。RNS衰减值检测19.853与空白组0.064比较有明显升高;血清AchR表达水平4.044与空白组30.746比较明显降低,具有明显统计学意义。3.应用“温阳补气”针法和溴吡斯的明治疗后,“温阳补气”针法组及溴吡斯的明组同模型组大鼠比较,大鼠Lennon症状评分降低,体重明显增加,RNS衰减值显著降低,血清AchR表达水平明显升高。4.在体重增加方面,“温阳补气”针法组结果明显高于溴吡斯的明治疗组(p<0.05);RNS衰减值和血清AchR表达水平两组之间无明显差异。
结论:1.本实验应用AchR片段129～145加上完全弗氏佐剂(CFA)免疫大鼠,结果实验大鼠与人类MG的临床症状改变相似,Lennon的评分标准评价症状等级、RNS衰减值和血清AchR-ab表达水平初步判定EAMG模型建立成功。2.“温阳补气”针法治疗EAMG有明显疗效。能够使EAMG大鼠体重明显增加;Lennon症状评分明显降低,肌力明显增加;RNS衰减值显著降低;血清AchR表达水平明显升高。3.治疗第14天后,“温阳补气”针法组与溴吡斯的明组疗效相当,无明显差异(p>0.05)。但从EAMG大鼠体重变化上比较,“温阳补气”针法组大鼠体重优于溴吡斯的明组。
Purpose: To establish AchR fragment of 129 to 145 classic animal model of myasthenia gravis - experimental autoimmune myasthenia gravis (Experimental autoimmune myasthenia gravis-EAMG) in rats, through the“wen yang bu qi”acupuncture therapy for low frequency repetitive EAMG rats electrical nerve stimulation (repetitive nerve stimulation,RNS) and serum acetylcholine receptor (Acetylcholine receptors AchR) of the study on the“wen yang bu qi”acupuncture therapy treatment of EAMG rats and its mechanism. In the treatment of atrophy and further enriches the theory,for the treatment of autoimmune diseases and the mechanism of providing a new tool.
Methods: Lewis rats were selected,the AchR fragment of 129 to 145 ratio of 1:2 emulsion with CFA fully established rat model of EAMG. The antigen emulsion to 200μl / only four foot pad injected rats Ministry (50μl),the amount of antigen to 50μg / only. After the first immunization,respectively,4 weeks,8 weeks,again with the method of immunization. Recorded body weight of rats,the reference standard evaluation Lennon symptoms symptom scores level,RNS attenuation expression and serum levels of AchR-ab to determine whether the model successfully. Successful model selected 30 rats were randomly divided into model group,pyridostigmine out group,“wen yang bu qi”acupuncture therapy group. Keeping with the conditions of the control group over the same period,arrests and treatment under the same conditions; model group is keeping the same conditions over the same period, not to any treatment;“wen yang bu qi”acupuncture therapy group was given“wen yang bu qi”acupuncture therapy group treatment ; pyridostigmine were given pyridostigmine Ming Ming treatment. Respectively on day 1 after onset and 14 days after the application of intervention Oxford-EMG evoked potential (Affiliated Hospital of Changchun University of Traditional Chinese Medicine) detection RNS attenuation change; enzymelinked immunosorbent assay (ELISA) detected the expression of AchR levels,and observe a week EAMG symptoms and weight changes in rats.
Results: 1. Application of 70 rats to model success 32 (after the success of Death 2), the success rate of 45.71%. 2.EAMG slow weight gain in rats,showing obvious symptoms of myasthenia gravis, clinical score 2.150, and the control group had significant difference (p <0.05). RNS attenuation test 19.853 and control group 0.064 was significantly higher compared; serum AchR expression 4.044 and the control group 30.746 decreased significantly compared with a statistically significant. 3. Application“wen yang bu qi”acupuncture therapy neostigmine and pyridostigmine treatment,“wen yang bu qi”acupuncture therapy group and the next group of pyridostigmine compared with model group,rats Lennon symptom score decreased body weight increased significantly,RNS attenuation value decreased significantly,Significantly serum levels of AchR expression. 4. In terms of weight gain,“wen yang bu qi”acupuncture therapy group was significantly higher than the result of Ming pyridostigmine treatment group (p <0.05); The weight of "yang qi"group is heavier then serum group.
Conclusion: 1. AchR fragment of the experimental application of 129 to 145 with complete Freund's adjuvant (CFA) immunization in rats,the results of experimental rat and human changes in clinical symptoms similar to MG,Lennon's rating criteria to evaluate symptoms of grade, RNS attenuation expression and serum levels of AchR-ab EAMG model to determine the initial success.2.“wen yang bu qi”acupuncture therapy treatment had significant effect on EAMG. Can make the weight of rats significantly increased EAMG; Lennon symptom score decreased,muscle strength increased significantly; RNS attenuation values were significantly reduced; significantly increased serum levels of AchR expression.3. 14 days of treatment,“wen yang bu qi”acupuncture therapy group and the next group of pyridostigmine efficacy rather, no significant difference (p> 0.05). However, body weight is relatively EAMG rats,“wen yang bu qi”acupuncture therapy group body weight than pyridostigmine next group,but not statistically significant.
方法:实验选取Lewis大鼠,将AchR片段129～145以1:2的比例与完全弗氏佐剂(CFA)完全乳化,建立EAMG大鼠模型。上述抗原乳剂以200μl/只注射于大鼠四足垫部(50μl),抗原量为50μg/只。分别于初次免疫后第4周、第8周,再次同法免疫接种。记录大鼠的体重变化,参考Lennon的评分标准评价模型症状等级,RNS衰减值和血清AchR-ab表达水平判定模型是否成功。选取造模成功大鼠30只,随机分为模型组、溴吡斯的明组、“温阳补气”针法组。空白组同期同条件饲养,相同条件下抓捕与处理;模型组大鼠也是同期同条件饲养,不予任何治疗;“温阳补气”针法组给予“温阳补气”针法组治疗;溴吡斯的明组给予溴吡斯的明治疗。分别于发病后的第1天及干预后第14天应用Oxford-肌电图诱发电位仪检测RNS衰减值变化;应用酶联免疫吸附法(ELISA法)检测AchR表达水平变化,并每周观察EAMG大鼠症状和体重变化。
结果:1.应用70只大鼠建立模型,成功32只(成功后死亡2只),成功率为45.71%。2.EAMG大鼠体重增加缓慢,表现出较为明显的肌无力症状,临床症状评分为2.150,与空白组比较有明显性差异(p<0.05)。RNS衰减值检测19.853与空白组0.064比较有明显升高;血清AchR表达水平4.044与空白组30.746比较明显降低,具有明显统计学意义。3.应用“温阳补气”针法和溴吡斯的明治疗后,“温阳补气”针法组及溴吡斯的明组同模型组大鼠比较,大鼠Lennon症状评分降低,体重明显增加,RNS衰减值显著降低,血清AchR表达水平明显升高。4.在体重增加方面,“温阳补气”针法组结果明显高于溴吡斯的明治疗组(p<0.05);RNS衰减值和血清AchR表达水平两组之间无明显差异。
结论:1.本实验应用AchR片段129～145加上完全弗氏佐剂(CFA)免疫大鼠,结果实验大鼠与人类MG的临床症状改变相似,Lennon的评分标准评价症状等级、RNS衰减值和血清AchR-ab表达水平初步判定EAMG模型建立成功。2.“温阳补气”针法治疗EAMG有明显疗效。能够使EAMG大鼠体重明显增加;Lennon症状评分明显降低,肌力明显增加;RNS衰减值显著降低;血清AchR表达水平明显升高。3.治疗第14天后,“温阳补气”针法组与溴吡斯的明组疗效相当,无明显差异(p>0.05)。但从EAMG大鼠体重变化上比较,“温阳补气”针法组大鼠体重优于溴吡斯的明组。
Purpose: To establish AchR fragment of 129 to 145 classic animal model of myasthenia gravis - experimental autoimmune myasthenia gravis (Experimental autoimmune myasthenia gravis-EAMG) in rats, through the“wen yang bu qi”acupuncture therapy for low frequency repetitive EAMG rats electrical nerve stimulation (repetitive nerve stimulation,RNS) and serum acetylcholine receptor (Acetylcholine receptors AchR) of the study on the“wen yang bu qi”acupuncture therapy treatment of EAMG rats and its mechanism. In the treatment of atrophy and further enriches the theory,for the treatment of autoimmune diseases and the mechanism of providing a new tool.
Methods: Lewis rats were selected,the AchR fragment of 129 to 145 ratio of 1:2 emulsion with CFA fully established rat model of EAMG. The antigen emulsion to 200μl / only four foot pad injected rats Ministry (50μl),the amount of antigen to 50μg / only. After the first immunization,respectively,4 weeks,8 weeks,again with the method of immunization. Recorded body weight of rats,the reference standard evaluation Lennon symptoms symptom scores level,RNS attenuation expression and serum levels of AchR-ab to determine whether the model successfully. Successful model selected 30 rats were randomly divided into model group,pyridostigmine out group,“wen yang bu qi”acupuncture therapy group. Keeping with the conditions of the control group over the same period,arrests and treatment under the same conditions; model group is keeping the same conditions over the same period, not to any treatment;“wen yang bu qi”acupuncture therapy group was given“wen yang bu qi”acupuncture therapy group treatment ; pyridostigmine were given pyridostigmine Ming Ming treatment. Respectively on day 1 after onset and 14 days after the application of intervention Oxford-EMG evoked potential (Affiliated Hospital of Changchun University of Traditional Chinese Medicine) detection RNS attenuation change; enzymelinked immunosorbent assay (ELISA) detected the expression of AchR levels,and observe a week EAMG symptoms and weight changes in rats.
Results: 1. Application of 70 rats to model success 32 (after the success of Death 2), the success rate of 45.71%. 2.EAMG slow weight gain in rats,showing obvious symptoms of myasthenia gravis, clinical score 2.150, and the control group had significant difference (p <0.05). RNS attenuation test 19.853 and control group 0.064 was significantly higher compared; serum AchR expression 4.044 and the control group 30.746 decreased significantly compared with a statistically significant. 3. Application“wen yang bu qi”acupuncture therapy neostigmine and pyridostigmine treatment,“wen yang bu qi”acupuncture therapy group and the next group of pyridostigmine compared with model group,rats Lennon symptom score decreased body weight increased significantly,RNS attenuation value decreased significantly,Significantly serum levels of AchR expression. 4. In terms of weight gain,“wen yang bu qi”acupuncture therapy group was significantly higher than the result of Ming pyridostigmine treatment group (p <0.05); The weight of "yang qi"group is heavier then serum group.
Conclusion: 1. AchR fragment of the experimental application of 129 to 145 with complete Freund's adjuvant (CFA) immunization in rats,the results of experimental rat and human changes in clinical symptoms similar to MG,Lennon's rating criteria to evaluate symptoms of grade, RNS attenuation expression and serum levels of AchR-ab EAMG model to determine the initial success.2.“wen yang bu qi”acupuncture therapy treatment had significant effect on EAMG. Can make the weight of rats significantly increased EAMG; Lennon symptom score decreased,muscle strength increased significantly; RNS attenuation values were significantly reduced; significantly increased serum levels of AchR expression.3. 14 days of treatment,“wen yang bu qi”acupuncture therapy group and the next group of pyridostigmine efficacy rather, no significant difference (p> 0.05). However, body weight is relatively EAMG rats,“wen yang bu qi”acupuncture therapy group body weight than pyridostigmine next group,but not statistically significant.
引文
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