“温阳补气”针法对EAMG大鼠IgG、IgM表达水平影响的研究
摘要
目的：乙酰胆碱受体（AChR）1129-145多肽片段与完全弗氏佐剂（CFA）乳化主动免疫Lewis大鼠建立实验性自身免疫性重症肌无力（EAMG）动物模型。通过观察“温阳补气”针法及溴吡斯的明药物治疗对EAMG大鼠血清IgG、IgM表达水平的影响，探讨“温阳补气”针法治疗MG的作用机制及发病机制，进一步丰富中医临床针灸治疗MG的理论依据，从而为临床中“痿证”的治疗提供了新的途径。
方法：选用雌性Lewis大鼠70只，随机抽取10只为空白对照组。60只大鼠以1:2的比例将乙酰胆碱受体（AChR）1129-145多肽片段和完全弗氏佐剂（CFA）完全乳化，分别注射于背部、尾根部、足垫部复制EAMG模型。每天观察大鼠肌无力症状，以Lennon等的分级法判定肌无力严重程度，以AChR-ab表达水平判定大鼠是否造模成功。将成功的EAMG大鼠31只，随机选取30只分为模型组、针灸治疗组（“温阳补气”针法）及药物治疗组（溴吡斯的明），每组各10只。空白对照组、模型组对照组在同等条件下，不做任何特殊处理；针灸治疗组针刺所选穴位后平补平泻，针上加灸（艾柱高约1cm，灸3壮），每天1次，每次30min，7天为一疗程，连续治疗两个疗程，疗程间休息1天；药物治疗组进行溴吡斯的明组灌胃给药，连续治疗15天。治疗结束后，4组大鼠均尾根部静脉采血，采用ELISA法检测各组大鼠血清中IgG、IgM表达水平。
结果：①60只大鼠进行EAMG模型复制，结果免疫4周后大鼠发病，出现活动减少、动作减缓、挣扎力量减弱、叫声降低、毛发变暗等症；加强免疫后大鼠肌无力症状逐渐加重，至造模结束时大鼠因MG症状过重死亡3只，ELISA法检测AChR-ab滴度，P/N比值均≥2.1，符合当今有关文献报道，成功制备31只EAMG大鼠，成功率约为51.7%。②各组治疗14天后，采用ELISA法检测各组大鼠血清中IgG、IgM表达水平，结果显示：针灸治疗组和药物治疗组EAMG大鼠血清中IgG表达水平均明显下降，针灸治疗组和药物治疗组分别与模型对照组比较，均具有极显著性差异(P<0.01)，但针灸治疗组疗效与药物治疗组相比，无显著性差异(P>0.05)；而IgM表达水平各组比较均无显著性差异(P>0.05)。
结论：①乙酰胆碱受体（AChR）1129-145多肽片段和完全弗氏佐剂（CFA）完全乳化主动免疫大鼠建立EAMG动物模型成功率较高。②“温阳补气”针法降低了EAMG大鼠血清中IgG的表达水平，影响自身免疫系统的应答反应，从而改善大鼠肌无力症状，对MG有一定的治疗作用，说明IgG表达水平与MG的发病机制关系密切。③“温阳补气”针法对EAMG大鼠血清中IgM表达水平无调节作用，说明IgM表达水平可能与MG的发病机制无紧密联系。
Purpose: To establish the rats model of experimental autoimmune myasthenia g ravis(Experimental autoimmune myasthenia gravis-EAMG) by emulsification liquid with AchRfragmentα1129-145and CFA. Observing EAMG rats serum IgG and IgM expression levelto explore the mechanisms and pathogenesis of MG by “warming yang and nourishing qi”Acupuncture and drug treatment method for EAMG rats. Further enrich the theoretical basisof Chinese medicine clinical acupuncture treatment of MG, and thus provides a new way forthe clinical treatment of “paralysis card”.
Method:70female Lewis rats are choosed in this experiment, and randomly selected10rats to blank control group. The other60rats are used to establish The rats model ofexperimental autoimmune myasthenia gravis, respectively injected in the back, base of thetail, footpad according to the proportion of1:2with the emulsification liquid of AchRfragmentα1129-145and CFA. To observe the symptoms of myasthenia gravis in rats everyday, and decide whether the model is successful by the ways of gravis Lennon and theexpression level of AChR-ab titer. Then randomly selecting30rat and dividing rats intomodel group, acupuncture treatment group (“warming yang and nourishing qi” acupuncture)and drug treatment group (pyridostigmine),10rats in each group. blank control group andmodel group without any treatments under the same conditions; acupuncture treatment groupadopts normal reinforcement and normal reduction with needle and moxibustion one time for30min each day, and do continuously twe courses of the treatment which includes seven daysin one course, and between the courses to have one day of rest; the drug treatment group topour the pyridostigmine into stomach each day, do continuously15days. At the end of thetreatment, vein blood in the roots of all the rats tail were extracted and detected by ELISA inserum of IgG, IgM expression level.
Result:①60rats to be established EAMG model after immunization for four weeksappear reduced activity, step-down action, weakened and lowers truggle, lower sounds anddarken hair symptoms; gravis symptoms gradually increase of rats after intensive immunation,and leading to3rats were dead because of serious gravis symptoms in the end. all AChR-abtiters shows P/N≥2.1which is according with the literature reported now days. the success rate of EAMG model is about51.7%.②IgG, IgM expression level in serummeasured by ELISA14days after the treatment showed that: the acupuncture treatment groupand the drug treatment group of EAMG rats serum IgG expression levels were significantlydecreased, acupuncture treatment group and the treatment group and model control groupcomparison, both has a significant sex difference (P <0.01), but the acupuncture groupefficacy compared with medical therapy group, no significant with sex differences (P>0.05);while the IgM expression level was no significant difference in each group(P>0.05).
Conclusion:①The success rate of EAMG animal model is higher by acetylcholinereceptor (AChR)1129-145peptide fragments and complete Freund's adjuvant (CFA)completely emulsified active immunization of rats.②IgG expression level in EAMG ratserum were significantly increased and IgM expression level is no significant changes, whichindicated that the changes of IgG expression level plays an important role in the pathogenesisof EAMG, and the close relationship is infered between IgG expression level and thepathogenesis of MG.③“warming yang and nourishing qi” Acupuncture can reduce theexpression level of IgG in the serum of EAMG rats, and thereby improving the symptoms ofmyasthenia gravis. That indicates “warming yang and nourishing qi” Acupuncture plays animportant role in the changes of IgG expression level in rats serum, and has good therapeuticfor MG in clinical.
方法：选用雌性Lewis大鼠70只，随机抽取10只为空白对照组。60只大鼠以1:2的比例将乙酰胆碱受体（AChR）1129-145多肽片段和完全弗氏佐剂（CFA）完全乳化，分别注射于背部、尾根部、足垫部复制EAMG模型。每天观察大鼠肌无力症状，以Lennon等的分级法判定肌无力严重程度，以AChR-ab表达水平判定大鼠是否造模成功。将成功的EAMG大鼠31只，随机选取30只分为模型组、针灸治疗组（“温阳补气”针法）及药物治疗组（溴吡斯的明），每组各10只。空白对照组、模型组对照组在同等条件下，不做任何特殊处理；针灸治疗组针刺所选穴位后平补平泻，针上加灸（艾柱高约1cm，灸3壮），每天1次，每次30min，7天为一疗程，连续治疗两个疗程，疗程间休息1天；药物治疗组进行溴吡斯的明组灌胃给药，连续治疗15天。治疗结束后，4组大鼠均尾根部静脉采血，采用ELISA法检测各组大鼠血清中IgG、IgM表达水平。
结果：①60只大鼠进行EAMG模型复制，结果免疫4周后大鼠发病，出现活动减少、动作减缓、挣扎力量减弱、叫声降低、毛发变暗等症；加强免疫后大鼠肌无力症状逐渐加重，至造模结束时大鼠因MG症状过重死亡3只，ELISA法检测AChR-ab滴度，P/N比值均≥2.1，符合当今有关文献报道，成功制备31只EAMG大鼠，成功率约为51.7%。②各组治疗14天后，采用ELISA法检测各组大鼠血清中IgG、IgM表达水平，结果显示：针灸治疗组和药物治疗组EAMG大鼠血清中IgG表达水平均明显下降，针灸治疗组和药物治疗组分别与模型对照组比较，均具有极显著性差异(P<0.01)，但针灸治疗组疗效与药物治疗组相比，无显著性差异(P>0.05)；而IgM表达水平各组比较均无显著性差异(P>0.05)。
结论：①乙酰胆碱受体（AChR）1129-145多肽片段和完全弗氏佐剂（CFA）完全乳化主动免疫大鼠建立EAMG动物模型成功率较高。②“温阳补气”针法降低了EAMG大鼠血清中IgG的表达水平，影响自身免疫系统的应答反应，从而改善大鼠肌无力症状，对MG有一定的治疗作用，说明IgG表达水平与MG的发病机制关系密切。③“温阳补气”针法对EAMG大鼠血清中IgM表达水平无调节作用，说明IgM表达水平可能与MG的发病机制无紧密联系。
Purpose: To establish the rats model of experimental autoimmune myasthenia g ravis(Experimental autoimmune myasthenia gravis-EAMG) by emulsification liquid with AchRfragmentα1129-145and CFA. Observing EAMG rats serum IgG and IgM expression levelto explore the mechanisms and pathogenesis of MG by “warming yang and nourishing qi”Acupuncture and drug treatment method for EAMG rats. Further enrich the theoretical basisof Chinese medicine clinical acupuncture treatment of MG, and thus provides a new way forthe clinical treatment of “paralysis card”.
Method:70female Lewis rats are choosed in this experiment, and randomly selected10rats to blank control group. The other60rats are used to establish The rats model ofexperimental autoimmune myasthenia gravis, respectively injected in the back, base of thetail, footpad according to the proportion of1:2with the emulsification liquid of AchRfragmentα1129-145and CFA. To observe the symptoms of myasthenia gravis in rats everyday, and decide whether the model is successful by the ways of gravis Lennon and theexpression level of AChR-ab titer. Then randomly selecting30rat and dividing rats intomodel group, acupuncture treatment group (“warming yang and nourishing qi” acupuncture)and drug treatment group (pyridostigmine),10rats in each group. blank control group andmodel group without any treatments under the same conditions; acupuncture treatment groupadopts normal reinforcement and normal reduction with needle and moxibustion one time for30min each day, and do continuously twe courses of the treatment which includes seven daysin one course, and between the courses to have one day of rest; the drug treatment group topour the pyridostigmine into stomach each day, do continuously15days. At the end of thetreatment, vein blood in the roots of all the rats tail were extracted and detected by ELISA inserum of IgG, IgM expression level.
Result:①60rats to be established EAMG model after immunization for four weeksappear reduced activity, step-down action, weakened and lowers truggle, lower sounds anddarken hair symptoms; gravis symptoms gradually increase of rats after intensive immunation,and leading to3rats were dead because of serious gravis symptoms in the end. all AChR-abtiters shows P/N≥2.1which is according with the literature reported now days. the success rate of EAMG model is about51.7%.②IgG, IgM expression level in serummeasured by ELISA14days after the treatment showed that: the acupuncture treatment groupand the drug treatment group of EAMG rats serum IgG expression levels were significantlydecreased, acupuncture treatment group and the treatment group and model control groupcomparison, both has a significant sex difference (P <0.01), but the acupuncture groupefficacy compared with medical therapy group, no significant with sex differences (P>0.05);while the IgM expression level was no significant difference in each group(P>0.05).
Conclusion:①The success rate of EAMG animal model is higher by acetylcholinereceptor (AChR)1129-145peptide fragments and complete Freund's adjuvant (CFA)completely emulsified active immunization of rats.②IgG expression level in EAMG ratserum were significantly increased and IgM expression level is no significant changes, whichindicated that the changes of IgG expression level plays an important role in the pathogenesisof EAMG, and the close relationship is infered between IgG expression level and thepathogenesis of MG.③“warming yang and nourishing qi” Acupuncture can reduce theexpression level of IgG in the serum of EAMG rats, and thereby improving the symptoms ofmyasthenia gravis. That indicates “warming yang and nourishing qi” Acupuncture plays animportant role in the changes of IgG expression level in rats serum, and has good therapeuticfor MG in clinical.
引文
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