重症肌無力是由自身抗體在神經肌肉接頭特異性的結合乙酰膽鹼受體和肌肉特異性激酶引起的一種獲得性免疫性疾病。疾病的主要症狀是骨骼肌的軟弱無力和易疲勞性。這一症狀在運動後尤為顯著，休息後會有所緩解。重症肌無力在世界範圍的發病率是百萬分之三到三十。由於近年來患者的數量在不斷增加，重症肌無力引起了醫學界的廣泛關注。但是，目前的診斷和治療措施還不能完全滿足臨床病人的需要。在本課題研究中，我們希望運用代謝組學的手段建立一種新的更加有效可靠的方法用於重症肌無力的診斷。同時，我們希望在代謝物的水平上來闡釋強肌健力方（一種中藥復方）對重症肌無力的治療作用。 / 本研究所用樣本來自42個重症肌無力病人和16個健康志願者。樣本由廣州中醫藥大學第一附屬醫院於二零零七年到二零零八年收集所得。診斷後，病人每日口服一定劑量的強肌健力方接受治療，連續服藥兩個月。分別在服藥前和治療後對病人抽血採樣。進一步分離血清後，樣品進行質譜分析。多元統計學方法如主成分分析，正交偏最小二乘和正交偏最小二乘判別分析等用於質譜數據的分析。 / 通過和健康者比較分析，我們在重症肌無力病人的血液中找到142個顯著改變的離子。其中，14個離子得到鑒定，包括：γ-氨基丁酸，2-哌啶酸，鳥氨酸，5,8-十四碳二羧酸，精胺，己酰肉毒鹼，N-油酰基甘氨酸，鞘氨醇-1-磷酸，聯原膽酸，糞甾烷酸，植物鞘氨醇-1-磷酸，鵝去氧膽酸甘氨酸結合物，輔酶Q4和甘氨酸膽。基於以上142個離子建立的數學診斷模型在診斷重症肌無力時表現出很高的靈敏度和特異性，分別高達92.8%和83.3%。強肌健力方能夠逆轉由重症肌無力引起的特異性代謝變化，將病人體內被改變的代謝網絡恢復正常，特別是大部分的代謝標誌物在治療後都恢復到了相對正常水平，包括：γ-氨基丁酸，哌啶酸，鳥氨酸，5,8-十四碳二羧酸，精胺，己酰肉毒鹼，N-油酰甘氨酸，鞘氨醇-1-磷酸，聯原膽酸，輔酶Q4和甘氨酸膽。 / 本研究揭示了基於液質聯用的代謝組學方法適用於探索重症肌無力的代謝標誌物，並提供了一種可用於診斷重症肌無力的新方法。同時，本研究證實強肌健力方適用於重症肌無力的治療，且無明顯副作用。 / Myasthenia gravis (MG) is an acquired autoimmune disease caused by specific autoantibodies against acetylcholine receptors (AChRs) and muscle-specific kinase (MuSK) proteins at the neuromuscular junctions. The disease is characterized by weakness and fatigability of the voluntary muscles that gets worse with exertion and improves with rest. The global incidence rate of MG is about 3-30 cases per million per year. In recent years, the worldwide prevalence rate of MG is increasing as a result of increased awareness. However, current diagnostic measures and treatments are not conclusive and satisfactory for MG. In this study, a mass spectrometry-based metabolomic strategy was applied to develop a novel and reliable diagnostic measure for MG on the basis of metabolic analysis, and to explore the therapeutic effect of Qiangji Jianli Fang (QJF, a newly developed Chinese medicine formula) on MG at the metabolite level. / Total 42 MG patients (13 males and 29 females) and 16 volunteers (5 males and 11 females) were recruited at the First Affiliated Hospital of Guangzhou University of Chinese Medicine between March 2007 and March 2008. The patients took QJF once per day for 2 months. Peripheral blood from patients was collected at diagnosis and after 2-month treatment, respectively. Sera prepared from the blood samples were monitored by the liquid chromatography Fourier transform mass spectrometry (LC-FTMS). Mass spectral data were analyzed by multivariate statistical analyses, including principal component analysis (PCA), orthogonal partial least squares (OPLS), and orthogonal partial least squares discriminant analysis (OPLS-DA). / By comparing analysis with the healthy volunteers, 142 significantly changed ions from serum metabolic profile of MG patients were picked out as the potential biomarkers of MG. Among of them, 14 ions were temporarily identified. They were gamma-aminobutyric acid (GABA), pipecolic acid, ornithine, 5,8-tetradecadienoic acid, spermine, hexanoylcarnitine, N-oleoyl glycine, sphingosine-1-phosphate (S1P), bisnorcholic acid, coprocholic acid, phytosphingosine-1-P, chenodeoxycholylglycine, coenzyme Q4, and cholylglycine. The developed OPLS-DA diagnostic model based on the 142 special ions showed a high sensitivity (92.8%) and specificity (83.3%) in detecting MG. QJF showed a powerful action on MG by recovering the holistic serum metabolic profile from the disease level to the normal level. Especially, the levels of GABA, pipecolic acid, ornithine, 5,8-tetradecadienoic acid, spermine, hexanoylcarnitine, N-oleoyl glycine, S1P, bisnorcholic acid, coenzyme Q4, and cholylglycine in MG patients were regulated to a relatively normal level after QJF treatment. / My results first indicated that the LC-FTMS-based metabolomics was a useful tool in biomarkers exploration of MG, and it was potentially applicable as a new diagnostic approach for MG. Also, my results demonstrated that QJF was a good optional choice for the treatment of MG, with no reported side effects. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Lu, Yonghai. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 113-129). / Abstract also in Chinese. / Thesis committee --- p.i / Declaration --- p.ii / Abstract (in English) --- p.iii / Abstract (in Chinese) --- p.vi / Acknowledgements --- p.viii / Table of contents --- p.ix / Abbreviations --- p.xiv / List of Tables --- p.xviii / List of Figures --- p.xix / Chapter 1: Introduction --- p.1 / Chapter 1.1 --- Myasthenia gravis --- p.1 / Chapter 1.1.1 --- History --- p.1 / Chapter 1.1.2 --- Epidemiology --- p.2 / Chapter 1.1.3 --- Clinical features --- p.2 / Chapter 1.1.4 --- Clinical classification --- p.4 / Chapter 1.1.5 --- Pathophysiology --- p.5 / Chapter 1.1.6 --- Diagnosis --- p.9 / Chapter 1.1.6.1 --- Physical examination --- p.9 / Chapter 1.1.6.2 --- Blood test --- p.10 / Chapter 1.1.6.3 --- Electrodiagnostic test --- p.10 / Chapter 1.1.6.4 --- Edrophonium test --- p.11 / Chapter 1.1.6.5 --- Imaging --- p.11 / Chapter 1.1.6.6 --- Pulmonary function test --- p.11 / Chapter 1.1.7 --- Treatment --- p.12 / Chapter 1.1.7.1 --- Medication --- p.12 / Chapter 1.1.7.2 --- Thymectomy --- p.12 / Chapter 1.1.7.3 --- Plasmapheresis and intravenous immunoglobulin --- p.13 / Chapter 1.2 --- Qiangji Jianli Fang --- p.14 / Chapter 1.2.1 --- Huang qi --- p.15 / Chapter 1.2.2 --- Dang shen --- p.16 / Chapter 1.2.3 --- Bai shu --- p.16 / Chapter 1.2.4 --- Dang gui --- p.17 / Chapter 1.2.5 --- Sheng ma --- p.17 / Chapter 1.2.6 --- Chai hu --- p.18 / Chapter 1.2.7 --- Chen pi --- p.18 / Chapter 1.2.8 --- Gan cao --- p.19 / Chapter 1.3 --- Metabolomics --- p.19 / Chapter 1.3.1 --- What’s metabolomics? --- p.20 / Chapter 1.3.1.1 --- Metabolites --- p.20 / Chapter 1.3.1.2 --- Metabolome --- p.21 / Chapter 1.3.1.3 --- Two terms: metabolomics and metabonomics --- p.21 / Chapter 1.3.2 --- How metabolomics works? --- p.22 / Chapter 1.3.2.1 --- Sample preparation --- p.22 / Chapter 1.3.2.1.1 --- Quenching --- p.23 / Chapter 1.3.2.1.2 --- Separating metabolites --- p.24 / Chapter 1.3.2.1.3 --- Sample concentration --- p.24 / Chapter 1.3.2.2 --- Analytical technologies (Sample analysis) --- p.25 / Chapter 1.3.2.3 --- Data analysis --- p.26 / Chapter 1.3.2.4 --- Database --- p.28 / Chapter 1.3.3 --- Why metabolomics? --- p.29 / Chapter 1.3.4 --- Metabolomics for human diseases --- p.30 / Chapter 1.3.5 --- Metabolomics for Traditional Chinese Medicine --- p.32 / Chapter 1.4 --- Objectives and significances of the present study --- p.34 / Chapter Chapter 2 --- Metabolic biomarkers of myasthenia gravis --- p.36 / Chapter 2.1 --- Introduction --- p.36 / Chapter 2.2 --- Materials and methods --- p.40 / Chapter 2.2.1 --- Chemicals --- p.40 / Chapter 2.2.2 --- Patients --- p.40 / Chapter 2.2.3 --- Volunteers --- p.42 / Chapter 2.2.4 --- Blood collection --- p.43 / Chapter 2.2.5 --- QC samples --- p.43 / Chapter 2.2.6 --- Sample processing --- p.43 / Chapter 2.2.7 --- Liquid chromatography-mass spectrometry --- p.44 / Chapter 2.2.8 --- Data analysis --- p.45 / Chapter 2.2.9 --- Metabolite identification --- p.45 / Chapter 2.3 --- Results --- p.46 / Chapter 2.3.1 --- Method validation --- p.46 / Chapter 2.3.2 --- An overall comparative analysis between 28 patients and 10 volunteers --- p.48 / Chapter 2.3.3 --- Classification of MG --- p.53 / Chapter 2.3.4 --- Comparative analysis of the metabolic changes in early- and late-stage MG patients respectively --- p.54 / Chapter 2.3.5 --- Biomarker identification --- p.56 / Chapter 2.4 --- Discussion --- p.58 / Chapter 2.5 --- Conclusion --- p.63 / Chapter Chapter 3 --- A novel diagnostic approach for myasthenia gravis --- p.64 / Chapter 3.1 --- Introduction --- p.64 / Chapter 3.2 --- Materials and methods --- p.68 / Chapter 3.2.1 --- Chemicals --- p.68 / Chapter 3.2.2 --- Patients and Volunteers --- p.69 / Chapter 3.2.2.1 --- Training set for establishment of diagnostic model --- p.69 / Chapter 3.2.2.2 --- Test set for evaluation of diagnostic model --- p.69 / Chapter 3.2.3 --- QC samples --- p.70 / Chapter 3.2.4 --- Sample processing --- p.71 / Chapter 3.2.5 --- Chromatography --- p.71 / Chapter 3.2.6 --- Mass spectrometry --- p.72 / Chapter 3.2.7 --- Data analysis --- p.72 / Chapter 3.3 --- Results --- p.72 / Chapter 3.3.1 --- Method validation --- p.73 / Chapter 3.3.2 --- Alterations in serum metabolic profile under MG --- p.74 / Chapter 3.3.3 --- Prediction of MG based on biomarkers --- p.74 / Chapter 3.3.4 --- Establishment of diagnostic model on the basis of metabolic profile --- p.77 / Chapter 3.3.5 --- Prediction of MG with diagnostic model --- p.79 / Chapter 3.4 --- Discussion --- p.80 / Chapter 3.5 --- Conclusion --- p.83 / Chapter Chapter 4 --- Qiangji Jianli Fang treatment for myasthenia gravis --- p.84 / Chapter 4.1 --- Introduction --- p.84 / Chapter 4.2 --- Materials and methods --- p.88 / Chapter 4.2.1 --- Chemicals --- p.88 / Chapter 4.2.2 --- Herbs --- p.88 / Chapter 4.2.3 --- Participants --- p.88 / Chapter 4.2.4 --- QC samples --- p.90 / Chapter 4.2.5 --- Sample processing --- p.90 / Chapter 4.2.6 --- Liquid chromatography-mass spectrometry --- p.90 / Chapter 4.2.7 --- Data analysis --- p.91 / Chapter 4.3 --- Results --- p.91 / Chapter 4.3.1 --- Method validation --- p.91 / Chapter 4.3.2 --- Symptomatic examination after QJF treatment --- p.92 / Chapter 4.3.3 --- Holistic metabolic responses to QJF treatment --- p.93 / Chapter 4.3.4 --- MG biomarkers changes after QJF treatment --- p.95 / Chapter 4.3.5 --- Drug-related biomarkers of QJF --- p.97 / Chapter 4.4 --- Discussion --- p.100 / Chapter 4.5 --- Conclusion --- p.103 / Chapter Chapter 5 --- Conclusions --- p.104 / Chapter Chapter 6 --- Perspectives --- p.107 / Chapter 6.1 --- Experimental autoimmune myasthenia gravis model --- p.107 / Chapter 6.2 --- Chemical composition of Qiangji Jianli Fang --- p.111 / References --- p.113 / Appendices --- p.130

INTRODUÇÃO: A Miastenia gravis (MG) é uma desordem imunológica com antígenos alvos conhecidos, com produção de anticorpos contra o receptor nicotínico de acetilcolina, AAChR e Musk, na junção neuromuscular, dificultando a transmissão do impulso nervoso e provocando fadiga e fraqueza flutuantes na musculatura ocular, facial, dos membros e respiratória. A terapêutica sintomática com inibidores de acetilcolinesterase e a etiopatogênica como a timectomia, corticosteróides, agentes citostásticos e imunoglobulinas são utilizadas e indicadas em acordo com a incapacidade e gravidade clínicas. A qualidade de vida (QV) é uma ferramenta utilizada para quantificar a eficácia e a resposta às terapêuticas adotadas, avaliar a efetividade e custos econômicos de novas estratégias terapêuticas, contribuir para planejar e aplicar os recursos para a saúde na comunidade. OBJETIVO: Avaliar a influência da terapêutica na QV e evolução clínica dos pacientes com MG. CASUÍSTICA: Foram avaliados 51 pacientes com MG, 38 submetidos a tratamento conservador e 13 timectomizados. MÉTODOS: Os instrumentos genéricos quantitativos de QV como WHOQOL, qualidade de vida relacionada à saúde (QVRS) SF-36, e depressão (BDI) foram utilizados. ESTATÍSTICA: Empregou-se análise univariada com os testes de Wilcoxon, U-Mann-Whitney, Fisher e razão de chance para avaliar a evolução clínica, a QV e QVRS e depressão; análise multivariada para a caracterização dos dois grupos de terapêutica considerando a interação de múltiplos fatores. RESULTADOS: O grupo submetido à terapêutica conservadora foi constituído por 16 pacientes do sexo masculino e 22 do feminino, com média de idade de 33,71±2,76 anos; o grupo timectomizado foi constituído por 6 homens e 7 mulheres, média de idade de 32,23±4,16 anos. Os pacientes do grupo conservador apresentaram melhora clínica estatisticamente significante (p <0,05) dos aspectos clínicos, da QV, da QVRS e depressão. Os timectomizados apresentaram melhora estatisticamente significante dos aspectos físicos e psicológicos da QV e QVRS e depressão, e melhora clínica evidente e não significante (p = 0,06). A análise multivariável mostrou r=0,65 para comparação das áreas; Wilks? Lambda para analisar as distâncias, X2 = 22,67; gl = 7; p = 0,05; as medidas centrais mostraram média = -0,42 dp = 1,0 para o grupo conservador; e média = 1,22, dp = 1,0 para o grupo timectomizado. CONCLUSÕES: Nesta amostra e no período avaliado constatamos que: 1) Os pacientes submetidos ao tratamento conservador apresentaram diferenças significantes com melhora clínica acentuada, de QV, de QVRS e depressão; 2) os timectomizados apresentaram melhora clínica, da QV e QVRS em seus aspectos físicos e psicológicos, e efetiva e significante melhora dos índices de depressão; 3) a análise multivariada revelou que a timectomia produziu efeito benéfico e significativo na recuperação da saúde e bem estar nos pacientes. / INTRODUCTION: Myasthenia gravis (MG) is an immune mediated disease with production of antibodies against post-synaptic acetylcholine receptor of neuromuscular junctions (AAChR,Musk) and orders in nervous impulse transmission. The disease´s clinical characteristics include fatigability and fluctuating weakness of voluntary muscles. Acetylcholinesterase inhibitors, thymectomy, corticosteroids, cytostatic agents and immunoglobulin are widely used and are indicated according to the patient´s disability and severity. Clinical manifestations, Quality of life (Qol), health-related (HRQol) analysis are used to evaluate response to therapy. Nowadays, Qol index is an important tool to evaluate the medical outcome, treatment efficacy, cost effectiveness and net benefit of new therapeutic strategies to determine whether their cost can be justified in the planning and application of health policies. OBJECTIVE: To evaluate the influence of conservative treatment and thymectomy on Qol and clinical response of myasthenic patients. SUBJECTS: Fifty-one myasthenic patients were chosen; 38 were submitted to conservative therapy and 13 to thymectomy. METHODS: Quantitative Qol tools such as WHOQOL, SF-36 and BDI were employed to evaluate Qol, HRQol and depression. STATISTICS: Univariate analysis by means of the Wilcoxon, U-Mann Whitney and Fisher tests, Chi-Square, odd ratio were used to follow the patient?s clinical status, evolution of Qol, HRQol and depression. Discriminant analysis was used to analyze the interation of multiple factors in the characterization of conservative and thymectomized groups. RESULTS: The conservative group of patients was constituted of 16 males and 22 females average age 33.71±2.76 years; the thymectomized group was composed of 6 males, 7 females, average age 32.23±4.16 years. Patients submitted to conservative therapy improved significantly in clinical progress, Qol, HRQol and depression. The follow-up of thymectomized patients showed a strong trend for clinical progress and significant improvement in physical and psychological Qol domains as well as in depression index. Discriminant analysis showed r = 0.65, p <0.05; Wilk?s Lambda X2 = 22.67, gl = 7; mean = -0.42, SD = 1.0 for conservative group; and mean = 1.22, SD = 1.0 for timectomized group. CONCLUSIONS: A prospective evaluation of a myasthenic patients sample revealed: 1) conservative treatment was found to have a strong and significant impact on clinical progress, Qol, HRQol and depression; 2) Thymectomy partly influenced Qol, specially physical and psychological aspects. There was also improvement in depression and clinical progress; 3) the evaluation of multiple parameters pointed to a strong and positive influence of thymectomy in the recovery of the patients.

INTRODUÇÃO: A Miastenia gravis (MG) é uma desordem imunológica com antígenos alvos conhecidos, com produção de anticorpos contra o receptor nicotínico de acetilcolina, AAChR e Musk, na junção neuromuscular, dificultando a transmissão do impulso nervoso e provocando fadiga e fraqueza flutuantes na musculatura ocular, facial, dos membros e respiratória. A terapêutica sintomática com inibidores de acetilcolinesterase e a etiopatogênica como a timectomia, corticosteróides, agentes citostásticos e imunoglobulinas são utilizadas e indicadas em acordo com a incapacidade e gravidade clínicas. A qualidade de vida (QV) é uma ferramenta utilizada para quantificar a eficácia e a resposta às terapêuticas adotadas, avaliar a efetividade e custos econômicos de novas estratégias terapêuticas, contribuir para planejar e aplicar os recursos para a saúde na comunidade. OBJETIVO: Avaliar a influência da terapêutica na QV e evolução clínica dos pacientes com MG. CASUÍSTICA: Foram avaliados 51 pacientes com MG, 38 submetidos a tratamento conservador e 13 timectomizados. MÉTODOS: Os instrumentos genéricos quantitativos de QV como WHOQOL, qualidade de vida relacionada à saúde (QVRS) SF-36, e depressão (BDI) foram utilizados. ESTATÍSTICA: Empregou-se análise univariada com os testes de Wilcoxon, U-Mann-Whitney, Fisher e razão de chance para avaliar a evolução clínica, a QV e QVRS e depressão; análise multivariada para a caracterização dos dois grupos de terapêutica considerando a interação de múltiplos fatores. RESULTADOS: O grupo submetido à terapêutica conservadora foi constituído por 16 pacientes do sexo masculino e 22 do feminino, com média de idade de 33,71±2,76 anos; o grupo timectomizado foi constituído por 6 homens e 7 mulheres, média de idade de 32,23±4,16 anos. Os pacientes do grupo conservador apresentaram melhora clínica estatisticamente significante (p <0,05) dos aspectos clínicos, da QV, da QVRS e depressão. Os timectomizados apresentaram melhora estatisticamente significante dos aspectos físicos e psicológicos da QV e QVRS e depressão, e melhora clínica evidente e não significante (p = 0,06). A análise multivariável mostrou r=0,65 para comparação das áreas; Wilks? Lambda para analisar as distâncias, X2 = 22,67; gl = 7; p = 0,05; as medidas centrais mostraram média = -0,42 dp = 1,0 para o grupo conservador; e média = 1,22, dp = 1,0 para o grupo timectomizado. CONCLUSÕES: Nesta amostra e no período avaliado constatamos que: 1) Os pacientes submetidos ao tratamento conservador apresentaram diferenças significantes com melhora clínica acentuada, de QV, de QVRS e depressão; 2) os timectomizados apresentaram melhora clínica, da QV e QVRS em seus aspectos físicos e psicológicos, e efetiva e significante melhora dos índices de depressão; 3) a análise multivariada revelou que a timectomia produziu efeito benéfico e significativo na recuperação da saúde e bem estar nos pacientes. / INTRODUCTION: Myasthenia gravis (MG) is an immune mediated disease with production of antibodies against post-synaptic acetylcholine receptor of neuromuscular junctions (AAChR,Musk) and orders in nervous impulse transmission. The disease´s clinical characteristics include fatigability and fluctuating weakness of voluntary muscles. Acetylcholinesterase inhibitors, thymectomy, corticosteroids, cytostatic agents and immunoglobulin are widely used and are indicated according to the patient´s disability and severity. Clinical manifestations, Quality of life (Qol), health-related (HRQol) analysis are used to evaluate response to therapy. Nowadays, Qol index is an important tool to evaluate the medical outcome, treatment efficacy, cost effectiveness and net benefit of new therapeutic strategies to determine whether their cost can be justified in the planning and application of health policies. OBJECTIVE: To evaluate the influence of conservative treatment and thymectomy on Qol and clinical response of myasthenic patients. SUBJECTS: Fifty-one myasthenic patients were chosen; 38 were submitted to conservative therapy and 13 to thymectomy. METHODS: Quantitative Qol tools such as WHOQOL, SF-36 and BDI were employed to evaluate Qol, HRQol and depression. STATISTICS: Univariate analysis by means of the Wilcoxon, U-Mann Whitney and Fisher tests, Chi-Square, odd ratio were used to follow the patient?s clinical status, evolution of Qol, HRQol and depression. Discriminant analysis was used to analyze the interation of multiple factors in the characterization of conservative and thymectomized groups. RESULTS: The conservative group of patients was constituted of 16 males and 22 females average age 33.71±2.76 years; the thymectomized group was composed of 6 males, 7 females, average age 32.23±4.16 years. Patients submitted to conservative therapy improved significantly in clinical progress, Qol, HRQol and depression. The follow-up of thymectomized patients showed a strong trend for clinical progress and significant improvement in physical and psychological Qol domains as well as in depression index. Discriminant analysis showed r = 0.65, p <0.05; Wilk?s Lambda X2 = 22.67, gl = 7; mean = -0.42, SD = 1.0 for conservative group; and mean = 1.22, SD = 1.0 for timectomized group. CONCLUSIONS: A prospective evaluation of a myasthenic patients sample revealed: 1) conservative treatment was found to have a strong and significant impact on clinical progress, Qol, HRQol and depression; 2) Thymectomy partly influenced Qol, specially physical and psychological aspects. There was also improvement in depression and clinical progress; 3) the evaluation of multiple parameters pointed to a strong and positive influence of thymectomy in the recovery of the patients.