本文關鍵詞： 蛋白質(zhì)芯片 蛋白質(zhì)組學 胃癌 血清標志物 早期診斷 預后 As2O3 己糖激酶　出處：《上海交通大學》2015年博士論文　論文類型：學位論文

【摘要】：隨著“人類基因組計劃”的完成和推進,生命科學研究已進入到系統(tǒng)生物學時代。蛋白質(zhì)組學作為一門迅速崛起的系統(tǒng)生物學分支,通過研究機體在不同生理病理狀態(tài)下動態(tài)變化的蛋白質(zhì)組成、表達水平與修飾狀態(tài),了解蛋白質(zhì)與蛋白質(zhì)之間的相互作用,來揭示蛋白質(zhì)的功能與細胞的活動規(guī)律,在癌癥研究領域展現(xiàn)出巨大的發(fā)展?jié)摿�。蛋白質(zhì)芯片技術作為蛋白質(zhì)組學研究的新興研究手段,以其高通量、并行、快速分析的優(yōu)勢在系統(tǒng)生物學研究中發(fā)揮著越來越重要的作用,必定能為癌癥的診斷和治療研究提供很好的技術支持,使人們能夠較為系統(tǒng)地研究在癌癥診斷和治療過程中蛋白質(zhì)的組成、變化規(guī)律。在本論文的研究中,我們利用蛋白質(zhì)芯片技術,圍繞胃癌診斷和治療相關的生物學問題,開展了尋找胃癌新型血清生物標志物和藥物治療靶點的研究。本論文首先將人蛋白質(zhì)組芯片技術運用到胃癌血清樣品的蛋白組學研究中,成功篩選出胃癌早期診斷和預后的血清生物標志物。通過對三組血清樣本(胃癌組、高危人群組和健康對照組)的樣品處理、芯片反應和數(shù)據(jù)分析,在不同血清樣本組間篩選出在三種抗體亞型(Ig G、IgM、Ig A)中存在顯著差異表達的自身抗體。通過更大規(guī)模的血清樣本在芯片上的驗證表明,我們篩選出44個在胃癌患者血清和健康人血清中顯著差異表達的自身抗體,其中4個在胃癌患者血清中的表達量顯著上調(diào)。我們對經(jīng)統(tǒng)計處理后排名最前的2個自身抗體,cops2和ctsf,采用免疫酶聯(lián)反應和免疫組化在血清水平和癌組織水平進行了詳盡的后期驗證工作。結(jié)果顯示,自身抗體cops2和ctsf在胃癌患者的血清中和蛋白質(zhì)cops2和ctsf在癌組織中的表達量跟正常對照組比較具有顯著差異。受試者工作特征曲線(roc)分析結(jié)果顯示,cops2和ctsf與目前臨床上使用的輔助胃癌診斷的生物標志物cea的roc曲線下面積分別為0.92、0.96和0.54,說明cops2和ctsf用于胃癌診斷的準確性明顯優(yōu)于cea;此外,cops2和ctsf同樣也能用于胃癌患者預后的判斷。上述結(jié)果均說明,cops2和ctsf是非常有潛力的胃癌早期診斷和預后判斷的血清標志物。接著,本論文將人蛋白質(zhì)組芯片技術引入到as2o3的藥物作用靶點探尋的研究中。as2o3治療急性早幼粒細胞白血病的療效和機理十分明確,但是對實體腫瘤的殺傷作用機制不是很清楚。我們在前期的研究中也發(fā)現(xiàn)as2o3能有效導致胃癌細胞凋亡,而誘導腫瘤細胞凋亡是藥物治療腫瘤最有效的方式�；诖�,我們利用人蛋白質(zhì)組芯片從系統(tǒng)水平進行as2o3直接相互作用蛋白質(zhì)的全局性發(fā)現(xiàn),篩選出與as2o3直接相互作用的一些關鍵蛋白質(zhì),期望從中能找到as2o3誘導胃癌細胞凋亡的具體作用機制。我們對篩選到的蛋白質(zhì)進行了體外、體內(nèi)及功能方面的驗證。并通過生物學功能分析,發(fā)現(xiàn)在這些差異表達的蛋白質(zhì)中,酶活性調(diào)節(jié)功能被顯著富集,主要集中在糖酵解途徑。我們進一步驗證了糖酵解途徑的重要限速酶己糖激酶ii在其中所發(fā)揮的作用。我們在選用人源胃癌細胞株sgc-7901基礎上,對細胞株進行己糖激酶ii過表達的改造,結(jié)果表明過表達的己糖激酶ii能顯著抑制as2o3對于胃癌細胞的殺傷作用,揭示了己糖激酶ii可以作為as2o3治療胃癌過程中重要的藥物作用靶點。最后,我們應用超高液相色譜/串聯(lián)質(zhì)譜(uplc/ms/ms)和氣相色譜質(zhì)譜聯(lián)用(GC/MS)平臺,對As2O3導致胃癌細胞凋亡的作用機制進行代謝組學方面的研究。我們通過對經(jīng)過As2O3處理6h、12h和24h的胃癌細胞株SGC-7901進行代謝產(chǎn)物的動態(tài)檢測分析,發(fā)現(xiàn)一部分代謝產(chǎn)物的變化集中在糖酵解途徑。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顯下降。除此之外,我們還檢測到As2O3可以影響線粒體的功能、炎癥和氧化應激反應以及抑制多胺的代謝等。代謝組學從系統(tǒng)層次對As2O3影響胃癌細胞的整體通路做了詳細的檢測和分析,得到的數(shù)據(jù)不僅僅與蛋白質(zhì)組學的數(shù)據(jù)相吻合,還找到了數(shù)個As2O3可能發(fā)揮作用的藥物作用靶點。由此可見,抑制糖酵解途徑可較有效地抑制胃癌細胞的增殖,同時As2O3可同時影響多個代謝通路的變化,可以從多方向多角度對胃癌的治療進行干預。綜上所述,本課題應用蛋白質(zhì)芯片技術對胃癌的診斷和治療進行研究。旨在從系統(tǒng)的角度出發(fā),嘗試篩選與胃癌診斷密切相關的血清候選標志物,以及治療胃癌的藥物作用靶點,為胃癌的臨床診斷、預后監(jiān)測、以及靶向藥物設計提供了思路。另外,本研究發(fā)展了一套尋找疾病血清生物標志物和藥物作用靶點的研究方法,為解決蛋白質(zhì)組學在腫瘤標志物和藥物作用靶點探尋研究中的技術難點提供了新的方法。
[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.

【學位授予單位】：上海交通大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R735.2
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本文編號：1533927