【作者】 颜淑敏；

【导师】 曾小峰； 张文； 李梦涛；

【作者基本信息】 中国协和医科大学 ， 内科学风湿免疫， 2008， 博士

【摘要】 课题一系统性血管炎血清蛋白质组学研究背景系统性血管炎由于临床表现复杂,病情偏重,且缺乏特异性生物学标志物,早期诊治困难,致残率、病死率高,预后差。因此,寻找系统性血管炎新的、特异性的生物学标志物已成为临床研究的热点。近年来,蛋白质指纹图谱技术即SELDI-TOF-MS技术已广泛用于对多种疾病,尤其是那些传统肿瘤标志物阴性的恶性肿瘤的早期诊断及生物标志物的筛查,并取得了成功,且后续对差异蛋白的鉴定成果业已见诸于报道。但其在系统性血管炎领域中的应用尚属空白。此外,本室前期研究显示抗膜突蛋白抗体与血管损伤、血管炎相关,推测其或许是系统性血管炎较特异的抗体。本研究首次在国内外采用蛋白质指纹图谱技术对白塞病、大动脉炎及ANCA相关性系统性血管炎(AASV)进行血清蛋白质组学研究,以期发现疾病特异性生物标志物及其组合模型,有助于对疾病的诊断、活动性监测及疗效评判。并对血清差异目的蛋白质分离富集的方法进行了初步探索,以期为后续差异蛋白质的鉴定提供技术支持。首次在国外内用免疫印迹法检测系统性血管炎患者血清抗膜突蛋白抗体的表达情况,探讨其临床意义。目的利用SELDI-TOF-MS技术检测系统性血管炎患者血清蛋白质指纹图谱,筛选出差异表达蛋白,构建系统性血管炎血清蛋白质指纹图谱分类树模型,寻找疾病活动性监测指标,分析差异蛋白与系统性血管炎临床表现、免疫学指标等临床资料的关系;如果能发现对系统性血管炎相对特异的差异蛋白质,则应用离子交换磁性微珠、SDS-PAGE,辅以SELDI-TOF-MS,对其进行分离和富集,利用MALDI-TOF-MS鉴定蛋白质。以重组膜突蛋白为抗原,采用免疫印迹法检测系统性血管炎患者血清抗膜突蛋白抗体的表达情况,探讨其临床意义。方法1.详细记录实验组及对照者的临床资料,并通过住院病历、门诊随诊记录对实验组进行随访。实验组为138例系统性血管炎患者,其中白塞病59例、大动脉炎42例及AASV 37例(包括韦格纳肉芽肿27例、显微镜下多血管炎9例及变应性肉芽肿性血管炎1例)。疾病对照组为30例狼疮性肾炎患者、15例冠心病患者。健康对照组为年龄、性别各相匹配的正常人,共115例。2.采用SELDI-TOF-MS技术检测所有入组者的血清蛋白质指纹图谱,应用生物信息技术,通过差异比对,筛查出差异表达蛋白质,构建系统性血管炎的疾病分类树模型、疾病活动性判定联合模式,寻找疾病特异性的血清标志物。3.分析差异表达蛋白与系统性血管炎病变分布、免疫学指标等临床资料的关系。4.采用离子交换磁性微珠、SDS-PAGE,辅以SELDI-TOF-MS分离富集差异表达的目的蛋白,并用MALDI-TOF-MS鉴定目的蛋白。5.利用重组膜突蛋白为抗原,采用免疫印迹法检测系统性血管炎患者血清中抗膜突蛋白抗体的阳性率,探讨其临床意义。结果1.由M/Z 7625.7、M/Z 3937.5和M/Z 12555.8所组成的白塞病诊断分类树模型、M/Z 7618.6组成的大动脉炎诊断分类树模型和M/Z 8337.8组成的AASV诊断分类树模型的敏感性依次是78.9%、91.7%、91.7%,特异性依次是80.0%、80.0%、100.0%;2.M/Z 7813.2峰强度≥1.20时,对白塞病活动性判断的敏感性为74.0%,特异性为77.8%;联合应用M/Z 8690.9、M/Z 16508.8和M/Z 4346.8三者对大动脉炎疾病活动性判断的敏感性为76.9%,特异性为100.0%;联合应用M/Z 11449.2、M/Z3276.6和M/Z 11671.9三者对AASV疾病活动性判断的敏感性为96.7%,特异性为100.0%;3.M/Z 13751.6峰强度≥0.64时,对白塞病内脏损害诊断的敏感性为84.2%,特异性为81.0%;4.M/Z 11689.7峰强度≥3.04时,对白塞病治疗无效者判断的敏感性为80.0%,特异性为87.5%;5.对蛋白沈脱液的蛋白质指纹图谱测定中可见,WCX磁性微珠捕获了血清目的蛋白M/Z 28.9kDa及M/Z 16.7kDa。SDS-PAGE电泳凝胶上可见相应两处条带。对28.9kDa处条带质谱鉴定结果为混合物。对16.7kDa处条带质谱鉴定结果为免疫球蛋白Kappa 1轻链;6.系统性血管炎患者血清抗膜突蛋白抗体的阳性率为32.6%,高于冠心病(6.7%)及正常人(5.0%),而与SLE患者(20%)相同;7.白塞病患者血清抗膜突蛋白抗体的阳性率为42.4%%,大动脉炎者为33.3%,AASV患者为16.2%;8.白塞病不同临床表现和实验室指标与血清抗膜突蛋白抗体无关;大动脉炎头臂动脉型患者抗膜突蛋白抗体的阳性率高于广泛型患者;9.抗膜突蛋白抗体阳性对系统性血管炎诊断的敏感性为32.6%,特异性为89.1%。结论1.蛋白质指纹图谱技术是一项极具潜力的适合于差异蛋白质组学研究的技术,可以用于对疾病生物标志物的筛查;2.由M/Z 7625.7、M/Z 3937.5和M/Z 12555.8所组成的白塞病分类树模型、M/Z7618.6组成的大动脉炎分类树模型和M/Z 8337.8组成的AASV分类树模型,对疾病的诊断有一定意义;3.M/Z 7813.2峰强度有助对白塞病疾病活动性的评判:M/Z 8690.9、M/Z 16508.8和M/Z 4346.8三者联合应用及M/Z 11449.2、M/Z 3276.6和M/Z 11671.9三者联合应用分别是评判大动脉炎、AASV疾病活动性的良好指标,均优于血沉、C反应蛋白等指标;4.M/Z 13751.6峰强度是白塞病内脏受累的良好标志物;5.M/Z 11689.7峰强度对评价白塞病患者对治疗的反应有一定的价值;6.所筛选出的系统性血管炎患者大量差异蛋白质可作为今后探索的目的蛋白;7.磁性微珠技术、蛋白质指纹图谱技术和Tricine-SDS-PAGE三者相结合来分离纯化蛋白质,具有良好的研究前景,是今后研究血清差异蛋白的有力武器。8.系统性血管炎患者血清中存在抗膜突蛋白抗体,其阳性率高于冠心病及正常人,而与SLE患者相同;不同系统性血管炎抗膜突蛋白抗体的阳性率不同,白塞病和大动脉炎患者明显高于AASV患者;白塞病不同临床表现和实验室指标与血清抗膜突蛋白抗体无关;大动脉炎头臂动脉型患者抗膜突蛋白抗体的阳性率高于广泛型患者;9.抗膜突蛋白抗体阳性对系统性血管炎的诊断具有一定意义。更多还原

【Abstract】 BackgroundSince nonspecific clinical manifestations, particularly the lack of specific biomarker, systemic vasculitis is a challenge for rheumatologists to diagnose early and has a poor prognosis with a higher morbidity and mortality. Currently, screening of specific and facilitative biomarkers has become a hot field in clinical studies on systemic vasculitis. During the last years, protein fingerprinting technology (also named as SELDI-TOF-MS) has been applied widely in medical field for the detection of diseases, especially for cancers without traditional tumor makers. An increasing number of cancer-related biomarkers for diagnosis, progression and prognosis have been identified successfully using SELDI-TOF-MS. When about systemic vasculitis, only one study on Wegener’s granulomatosis has been reported. On the other hand, in a previous study, we found the positive relation between anti-moesin antibody and the damage of vascular and vasculitis. So for the first time, we carry out studies on Behcet’s disease(BD), Takayasu arteritis(TA) and ANCA-associated systemic vasculitis (AASV) using SELDI-TOF-MS and detect the positive rate of anti-moesin antibody in patients with systemic vasculitis and evaluate its clinical significance.Objective1. To detect serum proteomic fingerprinting of patients with systemic vasculitis, and screen differentially expressed proteins, and establish classification tree models or serum biomarker pattern for diagnosis and monitoring on disease activity.2. To screen differential proteins and analyze their relationships with the clinical and immunological patterns of systemic vasculitis.3. To identify the aim proteins by the proteomics technique.4. To detect the positive rate of anti-moesin antibody in patients with systemic vasculitis, and analyze their clinical significance. Method1. Record the clinical dates of all subjects and follow up the experimental group with the clinical record in hospitalization and/or out-patient clinic. The experimental group is composed of 138 patients with systemic vasculitis, including 59 patients with BD, 42 with TA, 37 with AASV. 30 patients with lupus nephritis, 15 patients with coronary heart disease, 115 healthy persons were collected as controls. Serum samples of all subjects were collected when they entered the study.2. Detect all serum samples using SELDI-TOF-MS and obtain serum proteomic fingerprinting, and screen differentially expressed proteins using Ciphergen Biomarker Wizard, and establish classification tree models or serum biomarker pattern for diagnosis and monitoring on disease activity.using using Ciphergen Biomarker Wizard.3. Using Ciphergen Biomarker Wizard, screen differentially expressed proteins and analyze their relationships with the clinical and immunological patterns of systemic vasculitis.4. To identify the aim proteins by weak cation exchange interaction magnetic bead(WCX), SDS-PAGE, SELDI-TOF-MS and MALDI-TOF-MS.5. With recombinant moesin as antigen, sera were screened for the presence of anti-moesin antibody in all subjects by Western blotting and discuss its clinical significance.Results1. The diagnosis classification tree model for BD is composed of m/z 7625.7, m/z 3937.5 and m/z 12555.8 ions, while for TA and AASV are composed of m/z 7618.6 and m/z 8337.8 respectively, the sensitivities as follows respectively: 78.9%、91.7%、91.7%, and the specificities are as follows respectively 80.0%、80.0%、100.0%.2. The peak intensity of m/z 7813.2 gave 74.0% sensitivity and 77.8% specificity for active BD versus remission one at the cut-off point 1.20. The combination of m/z 8690.9、m/z 16508.8 and m/z 4346.8 has a sensitivity of 76.9% for active TA and a specificity of 100.0%, while the combination of m/z 11449.2、m/z 3276.6 and m/z 11671.9 has a sensitivity of 96.7% for active AASV and a specificity of 100.0%.3. The peak intensity of m/z 13751.6 has 84.2% sensitivity and 100.0% specificity for BD patients with systemic involvement at the cut-off point 0.64. 4. The peak intensity of m/z 11689.7 has 80.0% sensitivity and 87.5% specificity for BD patients failed to respond to therapy at the cut-off point 3.04.5. With SELDI-TOF-MS, the aim proteins m/z 28.9 and m/z 16.7 was purified and enriched by WCX magnetic bead, SDS-PAGE, and identified with MALDI-TOF-MS as mixture for the band 28.9kDa and immunoglobulin kappa 1 light chain for the band 16.7kDa respectively.6. Taking recombinant moesin as antigen, sera were screened for the presence of anti-moesin antibody in all subjects by Western blotting. The positive rates are follows: 32.6% (45/138) in systemic vasculitis, including 42.4% (25/59) in BD, 33.3% (14/42) in TA and 16.2% (6/37). 20.0% (4/20) in lupus nephritis, 6.7% (1/15) in coronary heart disease and 5.0% (1/20) in healthy persons.7. No relationship has been found between anti-moesin antibody and the clinical features of BD. While higher rate has been found in TA patients with the type of brachiocephalic artery involvememt versus those with the type of general involvememt.8. As a diagnosis method of systemic vasculitis, the sensitivity and specificity are 32.6% and 89.1% respectively.Conclusion1. Protein fingerprinting technology is a potential tool for discovery of novel biomarker in systemic vasculitis.2. The diagnosis classification tree models, including m/z 7625.7, m/z 3937.5 and m/z 12555.8 for BD, m/z 7618.6 for TA and m/z 8337.8 for AASV, have a certain value for diagnosis.3. Serum biomarker patterns, such as single ion m/z 7813.2 in BD, combination of m/z 8690.9、m/z 16508.8 and m/z 4346.8 in TA , and combination of m/z 11449.2、m/z 3276.6 and m/z 11671.9 in AASV, have significantly predictive value for active disease and better than ERS and CRP.4. Ion m/z13751.6 has a strong predictive value for systemic involvement in BD patients.5. Ion m/z11689.7 is a moderate predictive of patients with BD response to therapy.6. All of these differentially expressed proteins could be aimed as objects in the future studies. 7. Combination of WCX magnetic bead, Tricine-SDS-PAGE and SELDI-TOF-MS, would be a powerful arm to studies on serum differential proteome.8. The positive rate of anti-moesin antibody is higher in patients with systemic vasculitis than in those with coronary heart disease and healthy persons, and equal as patients in lupus nephritis. Among systemic vasculitis, patients with BD and TA have higher positive rate than those with AASV. Anti-moesin antibody has no relationship with the clinical features of BD.9. Anti-moesin antibody has moderate value for the diagnosis of systemic vasculitis.更多还原