蛋白质芯片技术在胃癌血清标志物及三氧化二砷作用靶点发现研究中的应用

发布时间：2018-02-25 09:46

  本文关键词： 蛋白质芯片 蛋白质组学 胃癌 血清标志物 早期诊断 预后 As2O3 己糖激酶　出处：《上海交通大学》2015年博士论文　论文类型：学位论文

【摘要】：随着“人类基因组计划”的完成和推进,生命科学研究已进入到系统生物学时代。蛋白质组学作为一门迅速崛起的系统生物学分支,通过研究机体在不同生理病理状态下动态变化的蛋白质组成、表达水平与修饰状态,了解蛋白质与蛋白质之间的相互作用,来揭示蛋白质的功能与细胞的活动规律,在癌症研究领域展现出巨大的发展潜力。蛋白质芯片技术作为蛋白质组学研究的新兴研究手段,以其高通量、并行、快速分析的优势在系统生物学研究中发挥着越来越重要的作用,必定能为癌症的诊断和治疗研究提供很好的技术支持,使人们能够较为系统地研究在癌症诊断和治疗过程中蛋白质的组成、变化规律。在本论文的研究中,我们利用蛋白质芯片技术,围绕胃癌诊断和治疗相关的生物学问题,开展了寻找胃癌新型血清生物标志物和药物治疗靶点的研究。本论文首先将人蛋白质组芯片技术运用到胃癌血清样品的蛋白组学研究中,成功筛选出胃癌早期诊断和预后的血清生物标志物。通过对三组血清样本(胃癌组、高危人群组和健康对照组)的样品处理、芯片反应和数据分析,在不同血清样本组间筛选出在三种抗体亚型(Ig G、IgM、Ig A)中存在显著差异表达的自身抗体。通过更大规模的血清样本在芯片上的验证表明,我们筛选出44个在胃癌患者血清和健康人血清中显著差异表达的自身抗体,其中4个在胃癌患者血清中的表达量显著上调。我们对经统计处理后排名最前的2个自身抗体,cops2和ctsf,采用免疫酶联反应和免疫组化在血清水平和癌组织水平进行了详尽的后期验证工作。结果显示,自身抗体cops2和ctsf在胃癌患者的血清中和蛋白质cops2和ctsf在癌组织中的表达量跟正常对照组比较具有显著差异。受试者工作特征曲线(roc)分析结果显示,cops2和ctsf与目前临床上使用的辅助胃癌诊断的生物标志物cea的roc曲线下面积分别为0.92、0.96和0.54,说明cops2和ctsf用于胃癌诊断的准确性明显优于cea;此外,cops2和ctsf同样也能用于胃癌患者预后的判断。上述结果均说明,cops2和ctsf是非常有潜力的胃癌早期诊断和预后判断的血清标志物。接着,本论文将人蛋白质组芯片技术引入到as2o3的药物作用靶点探寻的研究中。as2o3治疗急性早幼粒细胞白血病的疗效和机理十分明确,但是对实体肿瘤的杀伤作用机制不是很清楚。我们在前期的研究中也发现as2o3能有效导致胃癌细胞凋亡,而诱导肿瘤细胞凋亡是药物治疗肿瘤最有效的方式。基于此,我们利用人蛋白质组芯片从系统水平进行as2o3直接相互作用蛋白质的全局性发现,筛选出与as2o3直接相互作用的一些关键蛋白质,期望从中能找到as2o3诱导胃癌细胞凋亡的具体作用机制。我们对筛选到的蛋白质进行了体外、体内及功能方面的验证。并通过生物学功能分析,发现在这些差异表达的蛋白质中,酶活性调节功能被显著富集,主要集中在糖酵解途径。我们进一步验证了糖酵解途径的重要限速酶己糖激酶ii在其中所发挥的作用。我们在选用人源胃癌细胞株sgc-7901基础上,对细胞株进行己糖激酶ii过表达的改造,结果表明过表达的己糖激酶ii能显著抑制as2o3对于胃癌细胞的杀伤作用,揭示了己糖激酶ii可以作为as2o3治疗胃癌过程中重要的药物作用靶点。最后,我们应用超高液相色谱/串联质谱(uplc/ms/ms)和气相色谱质谱联用(GC/MS)平台,对As2O3导致胃癌细胞凋亡的作用机制进行代谢组学方面的研究。我们通过对经过As2O3处理6h、12h和24h的胃癌细胞株SGC-7901进行代谢产物的动态检测分析,发现一部分代谢产物的变化集中在糖酵解途径。其中6-磷酸葡萄糖、3-磷酸甘油醛、磷酸烯醇式丙酮酸以及乳酸的含量均明显下降。除此之外,我们还检测到As2O3可以影响线粒体的功能、炎症和氧化应激反应以及抑制多胺的代谢等。代谢组学从系统层次对As2O3影响胃癌细胞的整体通路做了详细的检测和分析,得到的数据不仅仅与蛋白质组学的数据相吻合,还找到了数个As2O3可能发挥作用的药物作用靶点。由此可见,抑制糖酵解途径可较有效地抑制胃癌细胞的增殖,同时As2O3可同时影响多个代谢通路的变化,可以从多方向多角度对胃癌的治疗进行干预。综上所述,本课题应用蛋白质芯片技术对胃癌的诊断和治疗进行研究。旨在从系统的角度出发,尝试筛选与胃癌诊断密切相关的血清候选标志物,以及治疗胃癌的药物作用靶点,为胃癌的临床诊断、预后监测、以及靶向药物设计提供了思路。另外,本研究发展了一套寻找疾病血清生物标志物和药物作用靶点的研究方法,为解决蛋白质组学在肿瘤标志物和药物作用靶点探寻研究中的技术难点提供了新的方法。
[Abstract]:With the "human genome project" and the completion of propulsion, life science research has entered into the era of system biology. Proteomics as a branch of the rapid rise of systems biology, through the study of the body in the dynamic change of different physiological and pathological state of the protein composition, expression and modification, the understanding of the interaction between protein and protein. To reveal the regularity and function of the cellular protein, showing great potential in the field of cancer research. New research in protein chip technology for proteomics research, with its high throughput, parallel, rapid analysis of the advantage of playing an increasingly important role in systems biology research, must be able to provide a good technical support for the research on the diagnosis and treatment of cancer, so that people can systematically study protein during the diagnosis and treatment of cancer Changes in the law of composition. In this paper, we use protein chip technology, focusing on the diagnosis and treatment of gastric cancer biology related issues, to carry out the search for research and therapeutic target of gastric cancer model serum biomarkers. The human proteome microarray technology is applied to the protein group in gastric cancer serum samples in the study, the success of screening serum biomarkers for diagnosis and prognosis of early gastric cancer. The serum samples of three groups (gastric cancer group, the high-risk group and healthy control group) sample processing, and data analysis of chip reaction in different groups, serum samples were screened in the three kinds of antibody subtypes (Ig G. IgM, Ig A) there was a significant difference in the expression of autoantibodies in serum samples. Through more extensive verification on the chip that we screened 44 in serum of gastric cancer patients and healthy human serum in difference table As one of 4 autoantibodies in patients with gastric cancer expression was up-regulated. The statistical treatment of the top 2 antibodies, cops2 and CTSF by ELISA and immunohistochemistry in detail later in serum and cancer tissue level verification work. Comparison showed that autoantibodies of cops2 and CTSF in serum of patients with gastric cancer and the expression of protein cops2 and CTSF in cancer tissues and normal control group with significant difference. The receiver operating characteristic curve (ROC) analysis showed that the area of ROC curve of cops2 and CTSF with adjuvant used in clinical diagnosis of gastric cancer biomarkers CEA under 0.92,0.96 and 0.54 respectively, indicating that cops2 and CTSF for gastric cancer diagnostic accuracy was significantly higher than that of CEA; in addition, cops2 and CTSF can also be used to judge the prognosis of gastric cancer patients. The results showed that, cops2 And CTSF is a potential serum marker for early gastric cancer diagnosis and prognosis. Then, this paper will study the curative effect and mechanism of.As2o3 protein chip technology is introduced into the drug targets As2O3 to explore in the treatment of acute promyelocytic leukemia is very clear, but the mechanism of cytotoxic effect on tumor is not very well. We also found in the previous study of As2O3 can effectively lead to the apoptosis of gastric cancer cells, and induce apoptosis of tumor cells is the most effective drug for the treatment of tumors. Based on this, we use the human proteome chip from the system level As2O3 direct interaction of global protein that screened key proteins directly with As2O3 the interaction of the specific mechanism from the expectations can be found in As2O3 induced apoptosis of gastric cancer cells. We screened the protein by in vitro, body Verify and function. And through biological function analysis, found that among these differentially expressed proteins, enzyme activity regulation function is significant enrichment, mainly concentrated in the glycolytic pathway. We further verify the glycolysis play important enzyme hexokinase II glycolytic pathway in the role of us in the selection. Human gastric cancer cell line SGC-7901 on the basis of cell line hexokinase II overexpression transformation, the results indicated that over expression of hexokinase II can significantly inhibit As2O3 cytotoxicity on gastric cancer cells, reveals the hexokinase II As2O3 can be used as a therapeutic drug targets important gastric cancer process. Finally, we used ultra high performance liquid chromatography / tandem mass spectrometry (uplc/ms/ms) and gas chromatography mass spectrometry (GC/MS) on the As2O3 platform, the research result of metabonomics of mechanism of apoptosis of gastric cancer cells. We After treated with As2O3 and 6h analysis, dynamic detection of 12h and 24h in gastric cancer cell line SGC-7901 was found to change metabolic products, some metabolites concentration in solution. The way glycolysis of glucose 6- phosphate and glyceraldehyde 3- phosphate content, phosphoenolpyruvate and lactic acid were significantly decreased. In addition, we also detected As2O3 can affect mitochondrial function, inflammation and oxidative stress and inhibition of polyamine metabolism. Metabonomics from the overall system pathway effect on As2O3 gastric cancer cell was detected and analyzed in detail, with the data obtained with not only the proteomics data, also found the drug action the target number of As2O3 may play an important role. Therefore, the inhibition of glycolysis can effectively inhibit the proliferation of gastric cancer cells, and As2O3 can influence many metabolic pathways, can To intervene from several directions for treatment of gastric cancer. In conclusion, diagnosis and treatment of the application of protein chip technology in gastric cancer. To start from the angle of system diagnosis and screening of gastric cancer serum to candidate closely related biomarkers, and drug treatment of gastric cancer targets for clinical diagnosis. The prognosis of gastric cancer and target monitoring, provides a way to design drugs. In addition, the research and development of a disease for serum biomarkers research methods and drug targets, in order to solve the problem of proteomics in tumor markers and drug targets to explore the technical difficulties of the study provides a new method.

【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.2
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本文编号：1533927