系统性红斑狼疮患者外周血临床蛋白质组学研究.pdfVIP

摘要 目的 分析系统性红斑狼疮患者外周血单个核细胞蛋白质表达谱的变化，建立和验证系统性 红斑狼疮患者血清蛋白指纹图谱诊断模型。 方法 1. 分别抽取系统性红斑狼疮患者及健康对照外周血，分离单个核细胞，抽提总蛋白， 进行双向电泳，凝胶银染显色，用ImageMaster 2D Platinum 5.0软件对获得的蛋白图谱进行 分析，寻找差异表达的蛋白质。利用基质辅助激光解吸电离飞行时间质谱 （MALDI-TOF-MS ）鉴定差异蛋白点。 2. 根据SLEDAI-2000评分标准把SLE病人分为活动期和稳定期两组，并选取RA疾病 对照组和健康对照组。分离血清，用弱阳离子交换磁珠（Dynabeads WCX ）纯化血清样本 中的多肽，然后通过MALDI-TOF质谱仪检测每个血清样本的纯化多肽谱，Clinprotools Software2.1分析组间差异多肽并建立诊断模型。 结果 1. 对照组凝胶蛋白点匹配率为71% ±4% ，病人组匹配率为72% ±4% 。对照组凝胶共 检出蛋白点791 ±17个，病人组检出781 ±17个。有11个蛋白点在SLE病人组表达上调，9 个表达下调，质谱分析共鉴定5个蛋白。以前的研究显示，我们鉴定的部分蛋白在SLE的发 病机制中起潜在的作用。 2. 组间差异多肽谱的分析结果显示，总共有30个多肽峰可以作为SLE病人的潜在标记 物，其中的6个在稳定期和活动期SLE之间的表达亦有差异。选取4个多肽峰应用遗传算法 建立SLE与正常对照之间的分类诊断模型，对样本做训练和交叉验证，其检测SLE的识别 率为92.57%，预测能力为85.25%。 结论 建立了 SLE 患者外周血单个核细胞二维电泳图谱，筛选并鉴定差异表达蛋白质；建 立了系统性红斑狼疮患者血清蛋白指纹图谱，筛选到差异多肽并建立了高敏感性和高特异 性的诊断模型。为从蛋白质组学角度探讨SLE 发病机制及早期诊断提供新思路和新依据。 关键词 红斑狼疮，系统性；双向电泳；磁珠；蛋白质组学 I Abstract Objective Our objective was to analyze the changes in the protein expression profile of peripheral blood mononuclear cells in systemic lupus erythematosus patients and to establish and validate the serum protein fingerprint model for the diagnosis of systemic lupus erythematosus. Methods 1. Peripheral blood was obtained from SLE patients and healthy controls, then mononuclear cells were isolated and total protein was extracted by one-step method. Two-dimensional gel electrophoresis was performed and gels were silver-stained. Protein maps were analyzed using ImageMaster 2D Platinum 5.0 software. Differentially expressed proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). 2. We divided SLE patients into two groups of active and stable states. RA patients were selected as di