基于SWATH-MS技术的肝癌定量蛋白质组学和激酶组学研究

发布时间：2018-04-24 11:25

  本文选题：蛋白质组 + 激酶组　； 参考：《中国人民解放军军事医学科学院》2017年博士论文

【摘要】：肝癌是世界范围内常见恶性肿瘤,在我国居癌症死因第二位,具有生存期短、死亡率高等特征。肝癌诊断标志物和药物靶标的发现仍是该研究领域亟待解决的难题。多个信号通路与肝癌的发生发展密切相关,且各信号通路之间存在相互关联,形成一个复杂网络。对整个信号通路网络蛋白质组和激酶组变化的研究有助于探索肝癌的发病机制,找到潜在的灵敏、特异性高的肝癌诊断标志物和药物靶标。在我国乙型肝炎病毒(Hepatitis B virus,HBV)感染是引发肝癌的最主要因素,本课题从定量蛋白质组和激酶组视角切入,主要关注对HBV相关肝癌发生发展起重要作用的蛋白质组和激酶组变化。采用SWATH-MS(按窗口顺序采集所有理论质谱谱图,Sequential window acquisition of all theoretical mass spectra)非标记定量方法和多种小分子抑制剂亲和富集激酶相结合的化学蛋白质组技术,对临床标本(肝癌及癌旁组织)蛋白质组和激酶组进行全景式定量表征,遴选表达量差异显著的蛋白质和激酶;进一步运用蛋白质印迹法进行临床样本确证,为探索肝癌发生发展的分子机制、发现潜在的肝癌诊断和药物靶向标志物提供研究线索。SWATH-MS技术是目前一种先进的非标记定量蛋白质组技术,可规避传统蛋白质组定量方法对于低丰度蛋白的定量偏差,具有样品用量少、样品前处理简单、蛋白鉴定覆盖率高和定量准确等优点,适合临床样本分析。本课题应用SWATH-MS技术对肝癌和对应癌旁组织的蛋白质组进行了比较研究,共定量4,216种蛋白质,其中338种蛋白质的表达量在肝癌和癌旁组织中有显著差异,包括191种蛋白质表达量在肝癌组织中增加,147种蛋白质表达量在肝癌组织中减少。对差异蛋白质的KEGG通路分析提示,表达量在肝癌组织中显著变化的蛋白质参与了多种不同的信号通路。本文重点对参与代谢通路的差异蛋白质进一步分析,结果表明,在肝癌组织中高表达蛋白质主要涉及糖酵解途径、磷酸戊糖途径和脂肪酸生物合成途径;在肝癌组织中低表达蛋白质主要涉及糖异生途径,丝氨酸、甘氨酸和肌氨酸生物合成/代谢和脂肪酸-氧化途径。这些代谢通路变化共涉及二十七种关键酶分子,包括PCK2、PDH和G6PD等。本研究定量的表达差异蛋白质将为肝癌药物靶标或诊断性标志物的发现提供重要线索。为定量研究肝癌组织样品中激酶的表达变化,本文运用化学蛋白质组技术,将丝氨酸/苏氨酸激酶抑制剂Purvalanol B和受体酪氨酸激酶抑制剂SU6668与EAH Sepharose 4B固相偶联,或将高效广谱激酶抑制剂VI16832与ECH Sepharose 4B固相偶联,富集激酶,然后结合非标记定量SWATH-MS技术,对肝癌组织与对应癌旁组织的激酶进行定量分析,共定量383种激酶,其中有93种激酶在肝癌和癌旁组织中表达量差异显著,包括80种表达量在肝癌组织中增加的激酶和13种表达量在肝癌组织中减少的激酶。93种差异显著激酶中有蛋白激酶73种,在人类激酶组9类激酶中均有分布。此外,本研究激酶组数据显示,表达量在肝癌组织中显著变化的激酶涉及了多条信号通路,主要包括MAPK信号通路、PI3K/Akt/m TOR信号通路、VEGF信号通路、Wnt信号通路和Hedgehog信号通路,VEGFR-2、CDK6和m TOR等参与了这些重要的信号通路。这些结果表明,多种小分子抑制剂亲和富集激酶结合SWATH-MS非标记定量的化学蛋白质组技术,能够有效的找到可能参与肝癌发生发展的关键激酶。此外,本课题对参与肝癌复杂信号通路网络的关键差异激酶及其小分子激酶抑制剂(Small-molecule kinase inhibitors,SMKIs)进行了文献调研。除已经被美国食品药品监督管理局(Food and drug administration,FDA)批准用于肝癌治疗含VEGFR-2靶点的多靶点小分子激酶抑制剂sorafenib外,以VEGFR-2为靶点用于肾细胞癌和甲状腺癌治疗的小分子激酶抑制剂lenvatinib,以及其他含VEGFR-2靶点的用于其他癌症治疗的多靶点小分子激酶抑制剂如pazopanib、vandetanib、axitinib、regorafenib、cabozantinib、ponatinib和nintedanib均有可能成为潜在的肝癌治疗药物。同时,以CDK4和CDK6为作用靶点用于乳腺癌治疗的小分子激酶抑制剂palbociclib和刚批准的ribociclib,以及以m TOR为靶点用于肾细胞癌治疗的小分子药物sirolimus、temsirolimus和everolimus,也均可能成为潜在的肝癌治疗药物。因此,应用小分子激酶抑制剂富集激酶和非标记定量SWAHT-MS技术相结合的化学蛋白质组学技术,开展肝癌差异激酶组的研究,不仅可为肝癌治疗药物靶标的发现提供基础,也可为探索肝癌生物机制和治疗提供新的策略。总之,本课题运用SWATH-MS技术对临床标本(肝癌及癌旁组织)蛋白质组和激酶组进行了全景式定量表征,定量了一批可能对肝癌发生发展起重要作用的差异蛋白质和激酶,为肝癌药物靶标或诊断性标志物的发现提供了重要数据,为探索肝癌治疗提供新的思路。
[Abstract]:Liver cancer is a common malignant tumor in the world. There are second causes of cancer death in China. It is characterized by short survival time and high mortality. The discovery of liver cancer markers and drug targets is still a difficult problem to be solved in this field. Multiple signal pathways are closely related to the development of liver cancer, and there is a mutual relationship between the various signal pathways. Association, forming a complex network. The study of the changes in the protein group and kinase group of the whole signaling pathway is helpful to explore the pathogenesis of liver cancer, and to find a potential sensitive, specific diagnostic marker and drug target for liver cancer. In our country, the Hepatitis B virus (HBV) infection is the most important factor in the cause of liver cancer. From the perspective of quantitative proteome and kinase group, this topic focuses on the changes in protein and kinase groups that play an important role in the development of HBV related liver cancer. SWATH-MS (Sequential window acquisition of all theoretical mass spectra) and a variety of non labeled quantitative methods are used. The chemical proteome technique of combining small molecule inhibitors with affinity enrichment kinase was used to determine the protein and kinase in the proteome and kinase group of the clinical specimens (liver cancer and para cancer tissue), and to select the protein and kinases with significant difference in expression, and further use the Western blot method to confirm the clinical samples to explore the development and development of liver cancer. The molecular mechanism, the discovery of potential liver cancer diagnosis and drug targeting markers.SWATH-MS technology is an advanced non labeling quantitative proteome technology, which can avoid the quantitative deviation of the traditional proteome quantitative methods for low abundance proteins, with less sample use, simple sample pretreatment, and protein identification coverage. It is suitable for clinical sample analysis with the advantages of high and quantitative accuracy. The SWATH-MS technique is used to compare the protein groups of the hepatocellular carcinoma and the adjacent tissues, and 4216 proteins are quantified. The expression of 338 proteins is significantly different in the liver and adjacent tissues, including 191 protein expressions in the liver cancer tissue In addition, 147 protein expressions are reduced in liver cancer tissues. KEGG pathway analysis of differential proteins suggests that proteins with significant changes in the expression of protein in HCC are involved in a variety of different signaling pathways. This article focuses on further analysis of the differential proteins involved in metabolic pathways, and the results indicate high expression in liver cancer tissues. Proteins are mainly involved in glycolysis pathway, pentose phosphate pathway and fatty acid biosynthesis pathway; low expression proteins in liver cancer mainly involve glycogen pathway, serine, glycine and serine biosynthesis / metabolism, and fatty acid oxidation pathways. These metabolic pathways involve twenty-seven key enzyme molecules, including PCK2, P DH and G6PD and so on. The quantitative expression of differential proteins in this study will provide important clues for the discovery of liver cancer drug targets or diagnostic markers. In order to quantify the changes in the expression of kinase in the samples of liver cancer tissue, this paper uses the chemical proteome technique to inhibit the serine / threonine kinase inhibitor Purvalanol B and receptor tyrosine kinase The solid phase coupling between SU6668 and EAH Sepharose 4B, or the solid phase coupling of the high efficient broad-spectrum kinase inhibitor VI16832 and ECH Sepharose 4B, enriching the kinase, and then combining with the unlabeled quantitative SWATH-MS technique, the quantitative analysis of the liver cancer tissues and the corresponding paracancerous kinases, and a total of 383 kinases, of which 93 kinds of kinases are in the liver and para cancer tissues. There were significant differences in the amount of expression, including the 80 kinds of kinases and 13 kinds of expressions of the 80 kinds of expression in the liver cancer tissue. The difference of the kinase.93 species in the liver cancer tissues was different in the significant kinase, which were distributed in the 9 kinases in the human kinase group. Furthermore, the number of kinases in this study showed that the expression was significantly changed in the liver cancer tissue. The kinase involves a number of signal pathways, including the MAPK signaling pathway, the PI3K/Akt/m TOR signaling pathway, the VEGF signaling pathway, the Wnt signaling pathway and the Hedgehog signaling pathway, and the VEGFR-2, CDK6, and m TOR. These results suggest that a variety of small sub inhibitors affinity enrichment kinase is combined with SWATH-MS unlabeled quantitative quantification. The chemical proteome technology can effectively find the key kinases that may be involved in the development of liver cancer. In addition, the topic has been investigated in the literature on key differential kinase and Small-molecule kinase inhibitors (SMKIs), which is involved in the complex signaling pathway of liver cancer. The Administration (Food and drug administration, FDA) approved the small molecular kinase inhibitor lenvatinib for the use of VEGFR-2 as a target for the treatment of renal cell carcinoma and thyroid cancer, as well as a small number of targets for other cancer treatments with VEGFR-2 as a target for the treatment of VEGFR-2 targets, the small molecular kinase inhibitor sorafenib containing VEGFR-2 targets. Molecular kinase inhibitors, such as pazopanib, vandetanib, axitinib, regorafenib, cabozantinib, ponatinib, and nintedanib, may be potential therapeutic drugs for liver cancer. At the same time, CDK4 and CDK6 are used as targets for small molecular kinase inhibitors, palbociclib and just approved ribociclib for the treatment of breast cancer, and targeted for M as targets. The small molecular drugs sirolimus, temsirolimus and everolimus for the treatment of renal cell carcinoma may also be potential therapeutic drugs for liver cancer. Therefore, the study of differential kinase group of liver cancer by combining small molecular kinase inhibitor enriching kinase and unlabeled quantitative SWAHT-MS technology can not only be used for the treatment of liver cancer, but also for the treatment of liver cancer. The discovery of drug targets provides a basis for the discovery of the biological mechanism and treatment of liver cancer. In conclusion, this subject uses SWATH-MS technology to quantify the quantitative characterization of the proteome and kinase groups of clinical specimens (liver cancer and adjacent tissues), and quantified a number of differential proteins that can play an important role in the development of liver cancer. Quality and kinase provide important data for the discovery of liver cancer drug targets or diagnostic markers, and provide new ideas for exploring the treatment of liver cancer.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.7
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本文编号：1796430