【作者】 李文辉；

【导师】 陈超； 周水珍； 宋义清；

【作者基本信息】 复旦大学 ， 儿科学， 2007， 博士

【摘要】 第一部分儿童重症肌无力血清学分型的临床研究目的：根据血清学对儿童重症肌无力(MG)分型，进行血清阳性重症肌无力(SPMG)和血清阴性重症肌无力(SNMG)临床相关问题的比较分析；在儿童MG中进行临床以及乙酰胆碱受体抗体(AchRab)、抗突触前膜受体抗体(PsmRab)和抗连接素抗体(Titinab)的随访，研究AchRab的变迁与临床状态之间的关系。方法：将我院2002年1月至2006年10月神经科病房收治的81例MG患儿进行自身抗体的检测，按血清型分型进行临床特征之间的比较(包括性别、临床分型、胸腺改变和重复电刺激阳性率)以及PsmRab、Titinab阳性率的比较。对儿童MG进行临床随访，比较不同血清学分型的复发和反复率；在临床状态发生改变时进行自身抗体的随访，了解儿童MG中自身抗体的变迁率以及不同临床状态下AchRab的变迁。结果：(1)一般情况：纳入研究的81例儿童MG中男41例，女40例，男女比为1：0.98；发病年龄中位数为3.03岁；就诊时病程平均为19.1±34.0月；Ⅰ型患儿63例，Ⅱ型患儿18例；2例女性患儿同时合并甲状腺功能亢进；2例有MG家族史。(2)SPMG和SNMG的比较：81例MG患儿中SPMG36例，SNMG45例：①AchRab、PsmRab和Titinab阳性率分别为44.4％、43.2％和21.7％。②Titinab阳性患儿无胸腺瘤的影像学依据。③SPMG和SNMG的性别、临床分型、胸腺影像学结果以及重复电刺激结果比较显示差异无统计学意义(P=0.585、1.000、0.783、0.162)；SPMG和SNMG的发病年龄比较差异有统计学意义(P＜0.05)，SPMG的发病年龄要早于SNMG；SPMG中的PsmRab和Titinab阳性率明显高于SNMG中阳性率，差异有统计学意义(P=0.000、0.003)。(3)随访结果：①临床随访：32例随访病例中有8例患儿出现了复发，8例患儿出现了反复，SPMG和SNMG的复发和反复率比较差异无统计学意义(P=0.154)；②自身抗体随访：AchRab、PsmRab和Titinab变迁率分别为25.0％、25.0％和19.2％；③AchRab变迁同临床状态之间的关系：伴随着病情缓解以及治疗结束时AchRab由阳性转变为阴性以及在病情反复时AchRab由阴性转变为阳性；在疾病缓解治疗结束时AchRab由阴性转变为阳性以及病情反复时AchRab由阳性转变为阴性，AchRab变迁不能代表临床状态的改变。结论：(1)近年来我院住院MG病例有增多趋势；本组MG患儿发病年龄小，主要集中于幼儿阶段；男女比例相当。(2)本组患儿中以SNMG居多，SPMG和SNMG的临床特征(包括性别、临床分型、胸部影像学以及重复电刺激结果)无差异，但SPMG的发病年龄同SNMG的发病年龄比较差异有统计学意义，SPMG的发病要早于SNMG。(3)多次进行AchRab检测、改进AchRab的检测技术，可能有助于正确进行血清学分型来指导临床。(4)本组患儿中PsmRab和Titinab同AchRab具有一定的相关性，Titinab阳性率明显低于成人且缺乏同胸腺瘤的关联。(5)不同血清学分型患儿在糖皮质激素治疗1年后都有复发，表明复发同糖皮质激素疗程问题值得探究。第二部分儿童重症肌无力血清学分型的动物实验研究目的：实验一通过儿童MG中初发MG(SNMG和SPMG)的血清被动转移小鼠，进行SPMG和SNMG血清被动转移动物模型的比较以及其发病机制的探讨；在实验一的基础上，实验二通过将糖皮质激素治疗不同时期的缓解MG的血清被动转移小鼠，进行糖皮质激素疗程问题的探讨。方法：(1)分组：选择43只健康、雌性、8周龄、清洁级C57BL／6小鼠，分为初发MG血清转移组(SPMG和SNMG)、糖皮质激素治疗MG缓解组(疗程3月、疗程6月和疗程12月)以及对照组(非MG和空白)。包括实验一分组：SPMG血清转移组、SNMG血清转移组、非MG对照血清转移组以及空白对照组；实验二分组：初发MG血清转移组、糖皮质激素治疗3月组、糖皮质激素治疗6月组、糖皮质激素治疗12月组、非MG对照血清转移组以及空白对照组。(2)实验方法：各组血清转移组将血清注射入小鼠腹腔0.5ml／天，连续7天；而空白对照组为相同条件下饲养7天。(3)观察指标：比较各组小鼠肌无力表现、肌电生理重复电刺激后衰减值，观察光镜下应用α-BGT-HRP定位突触后膜上AchR分布的变化以及电镜下突触结构的变化。结果：(1)实验一结果：①肌无力观察结果：与对照组小鼠相比，实验组小鼠包括(SPMG血清转移组和SNMG血清转移组)表现明显肌无力，主要为Ⅱ级和Ⅲ级改变。各组肌无力分级比较中，SPMG转移造模组、SNMG转移造模组分别同非MG对照血清转移组以及空白对照组比较显示差异有统计学意义(P＜0.01)，非MG对照血清转移组与空白对照组比较显示差异无统计学意义(P=0.05)，SPMG转移造模组与SNMG转移造模组比较显示差异无统计学意义(P=0.297)。②重复电刺激检查结果：对照组小鼠无衰减反应，SPMG血清转移组和SNMG血清转移组呈阳性衰减反应的88.9％和100％。各组重复电刺激衰减值比较中，SPMG转移造模组、SNMG转移造模组分别与非MG对照血清转移组以及空白对照组比较显示差异有统计学意义(P＜0.01)，非MG对照血清转移组与空白对照组比较显示差异无统计学意义(P=0.997)，SPMG转移造模组与SNMG转移造模组比较显示差异无统计学意义(P=0.911)。③光镜观察结果：空白对照组和非MG对照血清转移组中均显示棕黄色的AchR阳性沉积物以完整连续的细线条的形式锚在肌膜上，SPMG和SNMG血清转移造模小鼠的肌肉标本中棕黄色的AchR阳性沉积物变细，颜色变浅且棕黄色线条断断续续。④电镜观察结果：空白对照组和非MG对照血清转移组中突触结构(包括突触前膜、突触间隙以及突触后膜上的皱褶数量和形态)均正常，SPMG和SNMG患儿血清转移造模小鼠的肌肉标本中均可以见到突触结构的改变(包括突触间隙增宽，突触后膜上皱褶减少)以及突触前膜结构基本正常。(2)实验二结果：①肌无力观察结果：各组肌无力分级比较分析中，随着糖皮质激素治疗时间的延长，造模后小鼠的肌无力逐渐减轻；实验组小鼠分别与对照组小鼠比较显示差异有统计学意义(P＜0.05)；对照组小鼠组间比较显示差异无统计学意义(P=0.05)；实验组小鼠组间两两比较显示差异无统计学意义(P＞0.05)。②重复电刺激检查结果：随着糖皮质激素治疗时间的延长，造模后小鼠的衰减值的下降；衰减值比较分析中，实验组小鼠分别与对照组小鼠比较显示差异有统计学意义(P＜0.05)；对照组小鼠组间比较显示差异无统计学意义(P=-0.449)；实验组小鼠组间两两比较显示差异无统计学意义(P＞0.05)。③光镜观察结果：对照组中显示棕黄色的AchR阳性沉积物以完整连续的细线条的形式锚在肌膜上；实验组的各组肌肉标本表现为不同程度地棕黄色的AchR阳性沉积物变细、颜色变浅且沉积的线条断断续续。④电镜观察结果：对照组中突触前膜、突触间隙、突触后膜结构正常；而实验组各组中的标本中均可以见到不同程度的突触结构的改变(突触后膜上皱褶减少、变短)。结论：(1)雌性、8周龄的C57BL／6小鼠是建立被动转移血清的EAMG的易感动物。(2)儿童SPMG和SNMG血清被动转移后均能造成EAMG，且两组EAMG的肌无力和重复电刺激后衰减程度无差异，病理改变均为突触后膜上AchR的破坏。(3)儿童MG中SNMG的发病可能是由于直接和间接的对AchR的破坏导致的，关于SNMG血清中存在的对AchR间接破坏的致病因子将是我们进一步研究的方向。(4)儿童MG中糖皮质激素起到了重要的治疗作用，但是经过长疗程治疗的患儿血清转移至小鼠体内仍然能够产生EAMG，表明儿童MG可能仍需要长疗程的糖皮质激素治疗。更多还原

【Abstract】 PART 1 The clinical studies of myasthenia gravis inchildrenObjectives: Depending on having or not detectable AchR antibodies, myasthenia gravis were divided into seropositive myasthenia gravis and seronegative myasthenia gravis. In this part we compared the clinical problems of seropositive myasthenia gravis with seronegative myasthenia gravis. We did serological antibody tests during clinical follow-up and studied the correlation between the changes of serological classification and the clinical states.Methods: The data from 81 cases with myasthenia gravis treated in our hospital from 2002 to 2006 were analyzed. The levels of AchRab, PsmRab and Titinab in the sera were estimated. The clinical characteristics （including gender, the clinical classification, the image changes of thoracic gland and the positive results of repetitive nerve stimulation） were compared and the positive percents of PsmRab and Titinab between SPMG and SNMG were compared. During the clinical follow-up, the serological antibodies tests were done again when the clinical states had been changed. During the clinical follow-up, the differences of relapsed times between SPMG and SNMG were compared.Results: Eighty-one patients （41 were male and 40 were female）, onset median aged 3．03 years. According to the modified Osserman’s criteria, type I MG was found in 63 patients, typeⅡin 18．Two female patients had hyperthyroidism. Two patients had family histories of myasthenia gravis. According to the serological classification, SPMG was found in 36 patients and SNMG in 45．The positive percents of AchRab, PsmRab and Titinab were 44．4, 43．2 and 21．7．The Patients positive for Titinab had not thymoma in imaging. There were no differences in the clinical characteristers （including gender, the clinical classification, the image changes of thoracic gland and the positive results of Repetitive nerve stimulation） between SPMG and SNMG. There were significant differences in the percents of positiveness of PsmRab and Titinab between SPMG and SNMG. The positive percents of PsmRab and Titinab of SPMG were higher than SNMG. Thirty-two patients were followed up. The percents of changes of AchRab, PsmRab and Titinab were 25, 25 and 19．2．Eight patients relapsed after the therapies were finished and eight patients relapsed in the treatment. There were no relation between the clinical states and the transfer of AchRab.Conclusions: Recently the cases of myasthenia gravis admitted in our hospital had been increased. The onset ages were young. SNMG accounted for a considerable proportion of the cases. There were no differences in the clinical characteristics （including gender, the clinical classification, the image change of thoracic gland and the positive results of Repetitive nerve stimulation） between SPMG and SNMG. It may be helpful to make the correct serological classification for doctors that the sera of patients are estimated several times and the method of detecting the AchRab is improved. There were correlations among AchRab, PsmRab and Titinab. There was no correlation between positiveness of Titinab and thymoma. The percent of positiveness of Titinab was lower than those of adult reports. It is very worthy of studing of the correlation between relapses and the courses of treatment of glucocorticoid.PART 2 The animal experimental studies of myastheniagravis in childrenObjectives: We performed passive transfer studies using sera from the initial patients with myasthenia gravis including SPMG and SNMG and from the released patients who had taken glucocorticoid for different courses. We compared the groups of passive transfer models of SPMG and SNMG. We investigated the pathological mechanism of SPMG and SNMG. We investigated the appropriate course of treatment of glucocorticoid for MG.Methods: Passive transfer studies were performed using sera from the initial SPMG, the initial SNMG, the released patients who had taken glucocorticoid for different courses （three months, six months, twelve months） and non-MG control donors. The sera were injected intraperitoneally into female C57BL/6 mice （8 weeks） at a dose of 0．5ml/d for 7 consective days. On the seventh day from the start of injection, manifestation of muscle weakness was observed and classificated according on Lennon’s criteria. Twelve hours after the seventh day from the start of injection, electrophysiologic studies were performed and the amplitude of action potencial was recorded. The results of electrophysiology were demonstrated by D₅．α-BGT-HRP was used for the localization of the AchR in the endplate of mice. When electrophsiologic studies had been finished, mice were executed. Gastrocnemius muscles were obtained from the mice and then light microscope and electron microscope studies were performed. The blank control mice were bred in identical environment. Classification of muscle weakness, elecrophysiologic studies, light microscope studies and electron microscope studies were also performed in the blank control group.Results: Both the groups of passive transfer of the sera of SPMG and SNMG had significant muscle weakness, the control groups had no significant muscle weakness. There were significant differences in muscle weakness among the groups of SPMG, SNMG and the control groups. There were significant differences in D₅ among the groups of SPMG, SNMG and the control groups. There were no differences in muscle weakness and D₅ between the group of SPMG and the group of SNMG. The yellow lines of AchR deposition in sarcolemma were observed by the light microscope. The yellow lines of the group of SPMG and the group of SNMG were lighter and more discontinous than the lines of the non-MG control group and the blank control group. The construction of synapses of the group of SPMG was abnormal and the construction of synapses of the group of SNMG was also abnormal. Both the reductus of postsynaptic membranes of the group of SPMG and the group of SNMG were shorter and fewer than the control groups. The groups of the initial patients and released patients for taking glucocorticoid for different courses had significant muscle weakness, the control groups had no significant muscle weakness. There were significant differences of muscle weakness and D₅ among the groups of the initial patients, the groups of the released patients and the control groups. There were no differences of muscle weakness and D₅ among the initial groups and three released groups. The yellow lines of the initial groups and three released groups were lighter and more discontinous than the control groups. The constitution of synapses of the initial groups was abnormal and three released groups was abnormal too. The reductus of postsynaptic membrane of the initial groups and the released groups were shorter and fewer than the control groups.Conclusions: It was suitable to make the animal model through passive transfer of the sera of MG to female C57BL/6 mice （8 weeks）. There were no differences of muscle weakness and the decremental responses to repetitive nerve stimulation between the group of SPMG and the group of SNMG. Both the pathological changes of the group of SPMG and the pathological changes of the group of SNMG were the breakdown of AchR in postsynaptic membranes. The pathological mechanism of SNMG in childhood may be the direct or indirect breakdown of AchR in postsynaptic membranes. In the future we will focus on the pathological factors which result in the indirect breakdown of AchR in postsynaptic membrane. Glucocorticoid played a role on curing myasthenia gravis. Our data implied that the child patients of myasthenia gravis might need the longer courses of treatment of glucocorticoid.更多还原