【作者】 胡任飞；

【导师】 刘友章；

【作者基本信息】 广州中医药大学 ， 中医内科学， 2009， 博士

【摘要】 目的:重症肌无力是乙酰胆碱能运动神经元与骨骼肌之间突触功能障碍性疾病,其发病多归属于抗乙酰胆碱受体抗体的作用导致神经肌肉接头传递障碍。此外目前研究还发现,在重症肌无力的发病中往往可见肌细胞的损伤,如:线粒体的形态异常、呼吸链复合体-1缺失等等,无法用神经肌肉接头传递障碍进行简单地解释。另一方面,导师刘友章教授在临床上应用健脾祛湿方治疗重症肌无力取得了良好的疗效。因此本研究拟进行临床回顾性调查,总结重症肌无力诊治的现状,观察健脾益气方药对MG的影响及作用,并通过复制实验性自身免疫性重症肌无力动物模型,研究重症肌无力的肌细胞损伤的病理特点与其发病机制,对重症肌无力的发病机制进行补充,观察健脾祛湿方对其影响作用,探讨健脾祛湿方对防治重症肌无力的肌细胞损伤的作用机制,为中西医结合治疗重症肌无力的治疗方案提供理论指导。方法:本研究包括文献研究及实验研究。文献研究中,从祖国医学对重症肌无力的认识、现代医学对重症肌无力的研究、重症肌无力与肌细胞损伤的关系、重症肌无力模型的研究等四方面对重症肌无力的研究进行了详细的文献整理和总结。实验研究由两部分组成:第一部分是临床病历回顾研究,拟查阅广州中医药大学第一附属医院病案室近10年来重症肌无力的住院病历,调查分析重症肌无力住院病人的病因、病程、症状、诱因、并发症及治疗等,并进行统计学分析,观察健脾益气方药对重症肌无力的疗效,总结重症肌无力的诊治现状。第二部分是动物实验研究,选用SPF级Lewis大鼠45只,随机选取6只作为正常组(A组)正常饲养,其余39只以皮下注射人工合成的鼠源性乙酰胆碱α亚基97～116肽段来制作实验性自身免疫性重症肌无力动物模型,第6周末造模大鼠剩下36只,经肌电图检测后,确定成模32只,再将其随机分为模型对照组(B组)、强的松组(C组)、健脾祛湿方高剂量组(D组)、健脾祛湿方低剂量组(E组),每组各8只。成模2周后开始经灌胃药物干预,A、B组给予等容量生理盐水灌胃,C组给予强的松,剂量为5.4mg/kg体重·d,D、E组给予健脾祛湿方,剂量分别为37.6g/kg体重·d、18.8g/kg体重·d。以上各组灌胃液体量为10ml/kg体重,每日给药一次,其浓度及等效剂量按体重折算,共给药4周。实验结束后观察大鼠一般情况及体重、Lennon评分变化,观察肌肉线粒体及神经肌肉接头处超微结构的变化,并检测肌肉线粒体的DNA糖基化酶的表达、ATP含量、细胞色素氧化酶的活性及含量的改变。结果:1.临床回顾性研究:1.1重症肌无力住院患者男女比例1:1.23,发病高峰在31到50岁之间,大部分(72.73%)的病人病程在5年以下,分型以中度全身型为主;1.2积极预防控制感染,充分休息,避免劳累,规律服药,不擅自改变药量,饮食健康,调畅情志对MG病情的控制和恢复具有重要作用;1.3重症肌无力患者的舌脉以淡舌、白苔、细脉最多见,反映其主要病机为脾肾虚损,湿浊内停;1.4对重症肌无力患者中药使用频次分析表明:药物使用以补气健脾药为主,辅以利湿、补肾、活血、强筋药,符合对重症肌无力主要病机的分析。2.动物实验研究:2.1中药干预(健脾祛湿方)可改善实验性自身免疫性重症肌无力模型大鼠的肌无力症状、皮毛色泽;治疗前后体重比较,B组体重稍下降,C组略增加,但均无明显差异(P＞0.05),D、E组体重均较治疗前明显增加(P＜0.05),D、E组间比较无明显差异(P＜0.05)。2.2肌肉线粒体及神经肌肉接头处电镜观察:实验性自身免疫性重症肌无力模型大鼠存在肌纤维稀疏、紊乱,线粒体减少,神经肌肉接头处皱折局部紊乱、溶解、消失,突触前膜囊泡减少,线粒体等细胞器空泡化等超微结构改变,而中药干预(健脾祛湿方)能够改善上述病理改变,提示中药干预(健脾祛湿方)具有一定的减轻、改善实验性自身免疫性重症肌无力动物模型的肌细胞损伤作用。2.3 A组肌肉线粒体OGG1、NTH1、MPG的表达均最低,而B组OGG1、NTH1、MPG的表达均很高,与A组相比具有极显著差异(P＜0.01)。C组OGG1、NTH1、MPG的表达较B组极显著降低(P＜0.01),但仍极显著地高于A组(P＜0.01)。而D、E组OGG1、NTH1、MPG的表达明显升高,与其它三组均有极显著差异(P＜0.01),而D、E组间差异不明显(P＞0.05)。2.4 B组肌肉线粒体ATP含量明显降低,与A组相比具有极显著差异(P＜0.01);进行药物干预后,肌肉ATP含量均较B组明显提高(P＜0.05),但D、E组ATP含量均明显高于C组(P＜0.01),D组亦优于E组(P＜0.05)。2.5 B组肌肉线粒体CCO活性、含量均明显降低,与A组相比具有极显著差异(P＜0.01);进行药物干预后,C、D、E组肌肉线粒体CCO活性、含量均较B组明显提高(P＜0.05),但D、E组提高程度明显高于C组(P＜0.01),且D组亦优于E组(P＜0.01)。结论:1.皮下注射鼠源性乙酰胆碱受体α亚基97～116肽段可成功制作实验性自身免疫性重症肌无力大鼠模型。2.健脾祛湿方可改善实验性自身免疫性重症肌无力模型大鼠的肌无力症状、皮毛色泽,并能逐渐增加大鼠体重。3.健脾祛湿方具有在一定程度上的减轻、改善实验性自身免疫性重症肌无力模型大鼠肌肉线粒体及神经肌肉接头处病理改变的作用。4.健脾祛湿方干预能明显提高实验性自身免疫性重症肌无力模型大鼠肌肉线粒体DNA糖基化酶OGG1、NTH1、MPG的表达量。5.健脾清湿方干预能明显提高实验性自身免疫性重症肌无力模型大鼠肌肉线粒体ATP含量。6.健脾祛湿方干预能明显提高实验性自身免疫性重症肌无力模型大鼠肌肉线粒体CCO的活性及含量。更多还原

【Abstract】 Purposes:Myasthenia gravis is a synapse dysfunction disease between acetylcholine motorneuron and skeletal muscles.Its pathogenesy is due to acetylcholine antibodies which lead to the transmission block of neuromuscular junction. But nowaday some researches also discovered that there was muscle cell damage in myasthenia gravis,like paramorphia in mitochondria,missing of respiratory comples 1 et cetera,which could not simply be explained by the transmission block of neuromuscular junction.On the other side,my tutor-professor Liu Youzhang,applied Jianpi-qushi(JQ) decotion to treat myasthenia gravis and achieved satisfactory curative effect.Therefore this project is to carry out clinical retrospective investigation,to summarize the current situation of diagnoses and treatment of myasthenia gravis,to observe the effect of strengthening spleen and benefiting qi method on myasthenia gravis,and to research the pathology and pathogenesy of muscle cell damage in myasthenia gravis,filling up the pathogenesy of myasthenia gravis,to observe the effect of JQ decotion and discuss its curative effect on muscle cell damage of myasthenia gravis,to provide theory guidance for the combined treatment of Chinese medicine and western medicine.Methods:This project includes documentary research and experimental research.In documentary research,documents of myasthenia gravis were sorted and summarized in detail at four aspects,like knowledge of myasthenia gravis in Chinese medicine,researches on myasthenia gravis in mordern medicine,the relationship between myasthenia and muscle cell damage,researches on the models of myasthenia gravis.Experimental research consist of two parts.The first part is cases review on myasthenia gravis.The cases of myasthenia gravis in the near 10 years from the medical records department of the first affiliated hospital of Guangzhou University of Chinese medicine were to refered to.To analyse the causation,pathogenesis,symtoms,inducement,complication and treatment of myasthenia gravis.Then statistic analyses were to carry out. The curative effect of drugs of strengthening spleen and benefiting qi were to observed.The current situation of diagnoses and treatment of myasthenia gravis was to summarized.The second part was animal experimental research. 45 specific pathogen-free Lewis rats were chosen.6 of them were randomly selected into normal group(group A),which were raised normally.The rest of the rats were used to produce models of experimental autoimmune myasthenia gravis(EAMG) via subcutaneous injections of man-made synthetical peptide of acetylcholine receptorαsubunit 97～116.6 weeks later,36rats left.32 EAMG models were confirmed via electromyography,which were randomly divided into model control group(group B),prednisone group(group C),JQ decoction high dose group(group D),JQ decoction low dose group(group E).Each group contained 8 EAMG rats.2 weeks after EAMG was confirmed,drugs interference via intragastric administration began.Group C was given prednisone 5.4mg/kg body weight(BW)·d.Group D and E was given Jianpi-qingre-qushi decoction respective in the dose of 37.6mg/kg BW·d,18.8mg/kg BW·d.Group A and B were given the same amount of physiological saline.The amount of intragastric administration is 10ml/Kg BW,which was given once per day for 4 weeks.At the end of the experiment,general conditions,Lennon grade,ultrastructure of the muscle mitochondria and neuromuscular junction(NMJ) of the rats were observed. The DNA glycosylase expression,the amount of ATP,and the active and content of cytochrome oxidase(CCO) of the muscle mitochondria were detected.Results:1.Review on clinical study:1.1 In in-patient of myasthenia gravis,the male female ratio was 1:1.23.The morbidity peak lied between age 31 to 50.72.73%of the patients’ course were below 5 years.Most of them belonged to midrange generalized type.1.2 Active infection prevention and control,sufficient rest,avoidance of exertion,regular drug administration,never change drug dose personally,healthy diet,and agreeable emotion played a very important role in the control and recover of the patients’ condition.1.3 Most of the tongue and pulse presentations of myasthenia gravis were light tongue,white fur,and thready pulse,which reflected the main pathogenesis of myasthenia gravis that is spleen and kidney deficiency with turbid damp inside.1.4 The analyse of the frequency of Chinese drugs used in the treatment of myasthenia gravis showed:the most commonly used drugs was drugs that can invigorating qi and strengthening spleen.Other drugs included drugs invigorating kidney,drugs eliminating dampness,drugs activating blood circulation etc.This result accorded with the analyse of the main pathogenesis of myasthenis gravis.2.Animal experimental research:2.1 JQ decoction could improve the general conditions of EAMG rats,including symtoms,color and luster of their fur.The difference of BW before and after the administration was not significant between group B and C(P＞0.05).The BW of group D and E increased significantly after administration(P＜0.05),but the difference between group D and E was not significant(P＞0.05).2.2 Electron microscope observation of muscle mitochondria and neuromuscular junction:In EAMG rats,there were sparse mussy muscle fibre,decreasing mitochondria. And part of the winkle in NMJ were mussy,diffluent,disappeared,and uesicae in presynaptic membrane decreased,vacuolization in organelles like mitochondira existed.After administration of JQ decoction,the pathology changes mentioned above were improved,which showed JQ decoction could improve the MCD of EAMG.2.3 The expression of OGG1,NTH1,MPG of muscle mitochondria in group A was the lowest,and those in group B is very high.The difference between group A and B was significant(P＜0.01).The expression in group C decreased significantly than group B(P＜0.01),but still higher than group A(P＜0.01).The expression in group D and E were significantly higher than the other three groups(P＜0.01),but there was no significant difference between group D and E.2.4 The content of ATP of muscle mitochondria in group B decreased, significantly different from that in group A(P＜0.01).After the administration, the content in group C,D and E all increased,group D and E were higher than group C(P＜0.05),and group D were higher than group E(P＜0.01).2.5 The activity and content of CCO of muscle mitochondria in group B decreased, significantly different from that in group A(P＜0.01).After the administration, the activity and content in group C,D and E all increased.Group D and E were higher than group C(P＜0.05),and group D were higher than group E(P＜0.01).Conclusion:1.EAMG rat models could be established via subcutaneous injections of man-made synthetical peptide of acetylcholine receptorαsubunit 97～116.2.JQ decoction could improve the general Conditions of EAMG rats,including symtoms,color and luster of their fur,and their BW.3.JQ decoction could improve the pathology changes in muscle mitochondria and NMJ of the EAMG rats.4.JQ decoction could significantly improve the expression of OGG1,NTH1,MPG of muscle mitochondria in EAMG rats.5.JQ decoction could significantly improve the amount of ATP in muscle mitochondria of the EAMG rats.6.JQ decoction could significantly improve the activity and amount of CCO in muscle mitochondria of the EAMG rats.更多还原