【作者】 王建光；

【导师】 焦炳华；

【作者基本信息】 第二军医大学 ， 生物化学与分子生物学， 2011， 博士

【摘要】 类风湿关节炎(rheumatoid arthritis,RA)是一种复杂的自身免疫性疾病,以慢性滑膜炎为特点,逐渐导致关节软骨甚至骨的破坏及关节功能的障碍,该病表现出进展性及破坏性,极大影响患者的生活质量。尽管RA的病因仍不清楚,近年来研究显示活化的成纤维样滑膜细胞(FLS),作为复杂细胞调控网络中的一员,在RA的疾病发生、发展中发挥着重要作用,从而引起了人们的重视。但鉴于类风湿性关节炎在中晚期病变的发病机制具有复杂性的特点,单一信号转导通路的研究恐怕很难详尽地阐述其发病机制,因此急需对RA滑膜细胞内复杂的信号转导网络关系进行深入探讨。蛋白质组学作为一种分析表达蛋白质的主要技术,在过去的二十年里已经成为一项重要的发展领域。临床蛋白质组学的主要目的是在不同生理情况下,通过比较正常和疾病情况下的蛋白质组学资料,来鉴定该疾病的生物标志物,借此达到诊断或者治疗的目的。不断发展的高通量和基于敏感质谱分析的技术为检验各种生物样本的生理学和病理生理学提供了新机会。本课题的研究目的是用蛋白质组学的分析方法比较RA病人和正常人滑膜成纤维样细胞中的蛋白质表达差异来阐明类风湿性关节炎(RA)的发病机制。主要研究内容如下:1.采用shot-gun法lable-free方式进行类风湿性关节炎滑膜成纤维细胞的比较蛋白质组学研究:原代培养滑膜成纤维细胞,裂解后采用自动化2D-Nano-LC-ESI-MS/MS分析肽段,每组重复三次实验,根据大于一个unique peptide和p>0.05,在RA样品中鉴定到1060种蛋白质,在正常对照样品中鉴定到1292种蛋白质。2.差异蛋白质筛选:利用两样本t检验和蛋白质差异倍数方法筛选差异表达蛋白质。p<0.05,倍数>1.5的差异表达蛋白质有100个。RA中上调的蛋白质有46个,RA中下调的蛋白质有54个。3.随后进行系统的差异蛋白质的生物信息学分析GO分析结果:对筛选到的差异蛋白进行GO分析,GO数据库包含了基因参与的生物过程,所处的细胞位置,发挥的分子功能三方面信息,结果提示在RA滑膜成纤维细胞中血管生成相关蛋白质表达上调,氧化还原相关蛋白质表达下调。Pathway分析:对差异表达蛋白质进行KEGG pathway分析,搜索KEGG数据库。共找到65个相关的pathway,其中ECM-受体相互作用、三羧酸循环、乙醛酸二羧酸代谢、心肌收缩、色氨酸代谢、氨基酰- tRNA生物合成、膀胱癌、脂肪酸代谢、谷胱甘肽代谢、粘着斑、烟酸及烟酰胺代谢、磷酸戊糖途径、PPAR信号通路共14个pathway明显呈现蛋白质富集(p<0.05)。基因网络分析:同时整合3种不同的相互作用关系:1)KEGG数据库中基因之间的蛋白质相互作用、基因调控、蛋白质修饰等关系;2)已有的高通量实验,如酵母双杂交等证实的蛋白质-蛋白质相互作用;3)已有文献报道中提到的基因之间的相互作用,整合上述信息绘制基因网络图,网络中连接度高的基因,称为Hub基因。Hub基因往往对网络的稳定性起到重要作用,共找到与RA关系最为密切的四个Hub基因,它们是CTGF、THBS1、FN1和SOD2。4.在RA和正常滑膜组织中进行差异蛋白质的mRNA和蛋白质水平验证,结果与质谱鉴定结果一致,即THY-1、CTGF和THBS1表达上调,而SOD2表达下调。5.为了进一步确认血管形成相关因子THY-1, THBS1和CTGF的相互作用关系,我们进行了CTGF和THY-1的RNA干扰实验。结果显示在THY-1干扰下调之后,VEGF、CTGF和THBS1的mRNA和蛋白质水平表达都下调了。同时在CTGF干扰下调之后,VEGF的表达也下调了。然而,对照组的THBS1和THY-1的表达变化没有显著性差异。这些结果暗示在RA疾病发展进程中血管生成是一项重大事件,而THY-1是促进血管生成的一个上游调控分子。THY-1可以调控THBS1和CTGF的表达,进而影响VEGF的表达水平。6.个体验证RA滑液和血清中的四种Hub基因,发现CTGF、THBS1和FN1表达都有所上调,而SOD2含量有所下调,有望成为新的临床诊断指标。综上所述,在本次RA患者与正常人滑膜原代培养细胞的高通量比较蛋白质组学研究中,我们发现在RA患者中血管形成相关蛋白质表达上调,而氧化还原相关蛋白质的表达下调。这可能是血管翳形成的直接原因。血管形成的调节通路包括THY-1/ THBS1 /CTGF/ VEGF轴,该通路可能是促进RA患者的成纤维样滑膜细胞中血管形成的主要原因。更多还原

【Abstract】 Rheumatoid arthritis (rheumatoid arthritis, RA) is a complex autoimmune disease characterized by chronic synovitis, and gradually leads to the destruction of articular cartilage even the corruption of bone and the impairment of joints. The disease is often progressive and destructive; greatly affect the quality of life of patients. Although the etiology of RA remains unclear, recent studies have shown that activation of fibroblast-like synoviocytes (FLS), a complex network of cell regulation, play an important role in the pathological process of rheumatoid arthritis. These cells have been studied intensively, and significant progress has been made in elucidating the specific features of these fibroblasts. For it’s complex pathogenesis in advanced, a single signal transduction pathway will be difficult to elaborate in detail the pathogenesis, So we urgently need investigation into the complex signal transduction network in RA FLS.Proteomics, as a main technique for analysis of expressed proteins, has become an important developing area of research over the past two decades 1. A primary aim of clinical proteomics is the identification of biomarkers for diagnosis and therapeutic intervention of disease, by comparing proteomic profiles in normal and diseased conditions, and between different physiological states. The development of increasingly high-throughput and sensitive mass spectroscopy-based proteomic techniques provides new opportunities to examine the physiology and pathophysiology of many biological samples.To clarify the pathogenesis of rheumatoid arthritis (RA) by comparing protein expression in fibroblast-like synoviocytes (FLS) of RA patients and normal subjects using proteomics analysis.1. Using shot-gun method lable-free approach to do the Comparative Proteomics Analysis in rheumatoid arthritis synovial fibroblasts: Proteins extracted from primary cultured FLS of 50 RA patients and 10 normal subjects were analyzed by automated 2D-Nano-LC-ESI-MS/MS. According to the stringent criteria of having more than one unique peptide per protein present and a false discovery rate≤5%, 1060 proteins were identified from RA and 1292 proteins from normal subjects.2. Differentially expressed proteins were screened by 2-sample t-test (p<0.05) and fold change (fold>1.5), 100 differential proteins were screened out in RA. Of them, 46 proteins were up-regulated and the remaining 54 proteins were down-regulated. 3. Followed by a systematic difference bioinformatics analysis of proteins GO analysis: GO database contains genes involved in biological processes, cellular location, molecular function of three aspects of play information, results suggest that RA synovial fibroblasts in angiogenesis upregulation, redox-related protein was decreased.Pathway analysis: Searching KEGG database. We Found 65 related pathway, in which ECM-receptor interaction, the citric acid cycle, glyoxylate dicarboxylate metabolism, myocardial contraction, tryptophan metabolism, aminoacyl - tRNA biosynthesis, bladder cancer, fatty acid metabolism, Glutathione metabolism, focal adhesions, nicotinic acid and nicotinamide metabolism, pentose phosphate pathway, PPAR signaling pathway was presented (p <0.05).Gene network analysis: integration of information mapping the gene network diagrams, high network connective gene, known as the Hub genes, which often play an important role in the stability of the network, and the four Hub genes related to RA were found, which are CTGF, THBS1, FN1 and SOD2.4. Different proteins were validated in mRNA and protein levels of synovial tissue in RA and normal, the results were consistent with mass spectrometry, the expression of THY-1, CTGF, and THBS1 were increased, while SOD2 were downregulation.5. In order to further confirm the interaction between angiogenesis-related factor THY-1, THBS1 and CTGF, we downregulated CTGF and THY-1 by RNA interference experiments. The results showed that THY-1, VEGF, CTGF and THBS1 were downregulated after THY-1 knockdown, and VEGF and CTGF were downregulated after CTGF knockdown, and THY-1 is an upstream regulatory molecule in angiogenesis, which can regulate the expression of THBS1 and CTGF, thereby affecting the expression of VEGF.6. Individual verification of four Hub genes in synovial fluid and serum of RA, we found that the expression of CTGF, THBS1, and FN1 are increased, while SOD2 levels decreased.In concusion upregulation of vasculature development-related protein expression and downregulation of oxidation reduction-related protein expression in FLS are predominant factors contributing to the progression of RA.更多还原