本文選題：蛋白質(zhì)組 + 激酶組�。� 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2017年博士論文

【摘要】：肝癌是世界范圍內(nèi)常見惡性腫瘤,在我國居癌癥死因第二位,具有生存期短、死亡率高等特征。肝癌診斷標志物和藥物靶標的發(fā)現(xiàn)仍是該研究領(lǐng)域亟待解決的難題。多個信號通路與肝癌的發(fā)生發(fā)展密切相關(guān),且各信號通路之間存在相互關(guān)聯(lián),形成一個復(fù)雜網(wǎng)絡(luò)。對整個信號通路網(wǎng)絡(luò)蛋白質(zhì)組和激酶組變化的研究有助于探索肝癌的發(fā)病機制,找到潛在的靈敏、特異性高的肝癌診斷標志物和藥物靶標。在我國乙型肝炎病毒(Hepatitis B virus,HBV)感染是引發(fā)肝癌的最主要因素,本課題從定量蛋白質(zhì)組和激酶組視角切入,主要關(guān)注對HBV相關(guān)肝癌發(fā)生發(fā)展起重要作用的蛋白質(zhì)組和激酶組變化。采用SWATH-MS(按窗口順序采集所有理論質(zhì)譜譜圖,Sequential window acquisition of all theoretical mass spectra)非標記定量方法和多種小分子抑制劑親和富集激酶相結(jié)合的化學(xué)蛋白質(zhì)組技術(shù),對臨床標本(肝癌及癌旁組織)蛋白質(zhì)組和激酶組進行全景式定量表征,遴選表達量差異顯著的蛋白質(zhì)和激酶;進一步運用蛋白質(zhì)印跡法進行臨床樣本確證,為探索肝癌發(fā)生發(fā)展的分子機制、發(fā)現(xiàn)潛在的肝癌診斷和藥物靶向標志物提供研究線索。SWATH-MS技術(shù)是目前一種先進的非標記定量蛋白質(zhì)組技術(shù),可規(guī)避傳統(tǒng)蛋白質(zhì)組定量方法對于低豐度蛋白的定量偏差,具有樣品用量少、樣品前處理簡單、蛋白鑒定覆蓋率高和定量準確等優(yōu)點,適合臨床樣本分析。本課題應(yīng)用SWATH-MS技術(shù)對肝癌和對應(yīng)癌旁組織的蛋白質(zhì)組進行了比較研究,共定量4,216種蛋白質(zhì),其中338種蛋白質(zhì)的表達量在肝癌和癌旁組織中有顯著差異,包括191種蛋白質(zhì)表達量在肝癌組織中增加,147種蛋白質(zhì)表達量在肝癌組織中減少。對差異蛋白質(zhì)的KEGG通路分析提示,表達量在肝癌組織中顯著變化的蛋白質(zhì)參與了多種不同的信號通路。本文重點對參與代謝通路的差異蛋白質(zhì)進一步分析,結(jié)果表明,在肝癌組織中高表達蛋白質(zhì)主要涉及糖酵解途徑、磷酸戊糖途徑和脂肪酸生物合成途徑;在肝癌組織中低表達蛋白質(zhì)主要涉及糖異生途徑,絲氨酸、甘氨酸和肌氨酸生物合成/代謝和脂肪酸-氧化途徑。這些代謝通路變化共涉及二十七種關(guān)鍵酶分子,包括PCK2、PDH和G6PD等。本研究定量的表達差異蛋白質(zhì)將為肝癌藥物靶標或診斷性標志物的發(fā)現(xiàn)提供重要線索。為定量研究肝癌組織樣品中激酶的表達變化,本文運用化學(xué)蛋白質(zhì)組技術(shù),將絲氨酸/蘇氨酸激酶抑制劑Purvalanol B和受體酪氨酸激酶抑制劑SU6668與EAH Sepharose 4B固相偶聯(lián),或?qū)⒏咝V譜激酶抑制劑VI16832與ECH Sepharose 4B固相偶聯(lián),富集激酶,然后結(jié)合非標記定量SWATH-MS技術(shù),對肝癌組織與對應(yīng)癌旁組織的激酶進行定量分析,共定量383種激酶,其中有93種激酶在肝癌和癌旁組織中表達量差異顯著,包括80種表達量在肝癌組織中增加的激酶和13種表達量在肝癌組織中減少的激酶。93種差異顯著激酶中有蛋白激酶73種,在人類激酶組9類激酶中均有分布。此外,本研究激酶組數(shù)據(jù)顯示,表達量在肝癌組織中顯著變化的激酶涉及了多條信號通路,主要包括MAPK信號通路、PI3K/Akt/m TOR信號通路、VEGF信號通路、Wnt信號通路和Hedgehog信號通路,VEGFR-2、CDK6和m TOR等參與了這些重要的信號通路。這些結(jié)果表明,多種小分子抑制劑親和富集激酶結(jié)合SWATH-MS非標記定量的化學(xué)蛋白質(zhì)組技術(shù),能夠有效的找到可能參與肝癌發(fā)生發(fā)展的關(guān)鍵激酶。此外,本課題對參與肝癌復(fù)雜信號通路網(wǎng)絡(luò)的關(guān)鍵差異激酶及其小分子激酶抑制劑(Small-molecule kinase inhibitors,SMKIs)進行了文獻調(diào)研。除已經(jīng)被美國食品藥品監(jiān)督管理局(Food and drug administration,FDA)批準用于肝癌治療含VEGFR-2靶點的多靶點小分子激酶抑制劑sorafenib外,以VEGFR-2為靶點用于腎細胞癌和甲狀腺癌治療的小分子激酶抑制劑lenvatinib,以及其他含VEGFR-2靶點的用于其他癌癥治療的多靶點小分子激酶抑制劑如pazopanib、vandetanib、axitinib、regorafenib、cabozantinib、ponatinib和nintedanib均有可能成為潛在的肝癌治療藥物。同時,以CDK4和CDK6為作用靶點用于乳腺癌治療的小分子激酶抑制劑palbociclib和剛批準的ribociclib,以及以m TOR為靶點用于腎細胞癌治療的小分子藥物sirolimus、temsirolimus和everolimus,也均可能成為潛在的肝癌治療藥物。因此,應(yīng)用小分子激酶抑制劑富集激酶和非標記定量SWAHT-MS技術(shù)相結(jié)合的化學(xué)蛋白質(zhì)組學(xué)技術(shù),開展肝癌差異激酶組的研究,不僅可為肝癌治療藥物靶標的發(fā)現(xiàn)提供基礎(chǔ),也可為探索肝癌生物機制和治療提供新的策略。總之,本課題運用SWATH-MS技術(shù)對臨床標本(肝癌及癌旁組織)蛋白質(zhì)組和激酶組進行了全景式定量表征,定量了一批可能對肝癌發(fā)生發(fā)展起重要作用的差異蛋白質(zhì)和激酶,為肝癌藥物靶標或診斷性標志物的發(fā)現(xiàn)提供了重要數(shù)據(jù),為探索肝癌治療提供新的思路。
[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.

【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R735.7
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本文編號：1796430