本文選題：肝癌 + 索拉非尼　； 參考：《浙江大學(xué)》2016年博士論文

【摘要】：背景:肝癌為我國高發(fā)惡性腫瘤之一,每年約有38.3萬人死于肝癌,約占全球肝癌死亡總數(shù)的51%。目前肝癌治療手段有限,療效不理想,病死率極高,是困擾我國群眾健康的重大問題。肝癌的常見治療手段包括外科手術(shù)、肝動(dòng)脈栓塞化療術(shù)和消融術(shù)等,對(duì)于無法手術(shù)患者,化療也是重要手段之一。近年來新型分子靶向治療技術(shù)的發(fā)展為肝癌治療帶來了全新視野。靶向藥物索拉非尼可以通過抑制酪氨酸激酶阻止腫瘤新生血管的生成,還能經(jīng)Raf/MEK/ERK信號(hào)傳導(dǎo)通路直接抑制腫瘤生長。臨床試驗(yàn)表明索拉非尼在肝癌晚期患者中展現(xiàn)了良好的療效和耐受性,但存在耐藥現(xiàn)象,其耐藥機(jī)制尚不明確,一般認(rèn)為與腫瘤細(xì)胞、腫瘤基質(zhì)和個(gè)體遺傳耐藥性有關(guān)。相關(guān)研究表明肝癌耐藥相關(guān)分子的表達(dá)變化能指示患者療效敏感性,因此通過探索潛在生物標(biāo)志物,建立索拉非尼治療療效的分子生物標(biāo)志物譜,將有利于針對(duì)性給藥,實(shí)現(xiàn)精準(zhǔn)醫(yī)療;此外對(duì)于關(guān)鍵分子的機(jī)制研究,將解決耐藥發(fā)生,并推動(dòng)新藥研發(fā)。方法:1、外科(肝切除/肝移植)術(shù)后復(fù)發(fā)患者接受索拉非尼治療,根據(jù)影像學(xué)表現(xiàn)結(jié)合血清學(xué)指標(biāo)(AFP)將入組患者分為耐藥型和敏感型。取各組肝癌及癌旁組織,同時(shí)以其他肝硬化患者及健康供肝組織作為基準(zhǔn)對(duì)照進(jìn)行iTRAQ實(shí)驗(yàn),比較耐藥組與敏感組之間組織蛋白表達(dá)差異,進(jìn)而開展GO、KEGG等生物信息學(xué)分析,發(fā)現(xiàn)特征標(biāo)志物譜和所涉及的生物學(xué)通路。2、建立肝癌細(xì)胞體外索拉非尼耐藥模型,在模型中采用Western blot技術(shù)對(duì)特征標(biāo)志物的表達(dá)差異進(jìn)一步驗(yàn)證。3、利用耐藥模型,結(jié)合PPARy拮抗劑及激動(dòng)劑,采用CCK8法檢測PPARγ信號(hào)通路外在干預(yù)下肝癌細(xì)胞增殖情況的變化,細(xì)胞流式技術(shù)研究細(xì)胞周期阻滯和凋亡對(duì)肝癌細(xì)胞增殖抑制的作用,探索PPARγ拮抗劑潛在的抗索拉非尼耐藥價(jià)值。結(jié)果:1、利用iTRAQ技術(shù),發(fā)現(xiàn)索拉非尼敏感組和耐藥組表達(dá)差異達(dá)到1.5倍以上的蛋白有222個(gè),而表達(dá)差異達(dá)到2倍以上的蛋白57個(gè),其中表達(dá)上調(diào)蛋白有26個(gè),表達(dá)下調(diào)蛋白有31個(gè);2、GO分析表明差異蛋白多涉及小分子代謝過程、分解代謝過程、羧酸代謝過程及含氧酸的代謝過程,而KEGG的信號(hào)通路分析同樣表明,差異蛋白富集在糖代謝、脂肪酸代謝和糖酵解通路;3、利用體外耐藥模型對(duì)2倍以上差異蛋白的驗(yàn)證發(fā)現(xiàn)包括ITGA6、FN1、ICAM1、ITPA、BAX、TXNDC17和MSH2等蛋白差異與體內(nèi)結(jié)果一致,可作為機(jī)制研究的候選蛋白;4、基于生物信息學(xué)分析結(jié)果,發(fā)現(xiàn)PPARs特別是PPARγ在耐藥模型中表達(dá)發(fā)生顯著變化;5、發(fā)現(xiàn)PPARγ的拮抗劑T0070907能夠增強(qiáng)肝癌細(xì)胞對(duì)索拉非尼的敏感性,另一個(gè)拮抗劑GW9662未能達(dá)到同樣效果;6、,.拮抗劑T0070907及GW9662的聯(lián)用未能進(jìn)一步增強(qiáng)細(xì)胞對(duì)索拉非尼的敏感性;7、肝癌細(xì)胞對(duì)索拉非尼敏感性的增強(qiáng)是細(xì)胞周期阻滯所致。結(jié)論:1、通過比較索拉非尼耐藥和敏感患者組織蛋白表達(dá)譜,發(fā)現(xiàn)多個(gè)差異蛋白,涉及脂肪酸代謝等生物學(xué)過程及信號(hào)通路;2、PPARγ可能是肝癌索拉非尼耐藥發(fā)生的關(guān)鍵因子,其拮抗劑T0070907可增強(qiáng)肝癌細(xì)胞的敏感性,具有潛在的臨床應(yīng)用價(jià)值。
[Abstract]:Background: liver cancer is one of the high incidence of malignant tumors in China. About 383 thousand people die of liver cancer every year, accounting for about 51%. of the total number of cancer deaths in the world. At present, the treatment of liver cancer is limited, the curative effect is not ideal and the fatality rate is very high. It is a major problem perplexing the health of the masses in our country. The common treatment means of liver cancer include surgery, hepatic arterial chemoembolization and chemotherapy. In recent years, the development of new molecular targeting therapy has brought new field of vision for the treatment of liver cancer. The target drug Sola Fini can inhibit the formation of neovascularization of tumor by inhibiting tyrosine kinase, and can also suppress the swelling directly through the Raf/MEK/ERK signal transduction pathway. Tumor growth. Clinical trials show that Sola Fini has shown good efficacy and tolerance in advanced liver cancer patients, but there is a drug resistance phenomenon. The mechanism of drug resistance is not clear. It is generally considered to be related to tumor cells, tumor matrix and individual genetic resistance. Therefore, by exploring potential biomarkers and establishing molecular biomarker spectra of sorafenib treatment, it will be beneficial to targeted drug delivery and accurate medical treatment. In addition, the mechanism of key molecules will be studied to solve the drug resistance and promote new drug development. Methods: 1, surgery (hepatectomy / liver transplantation) for recurrent patients after surgery. In the treatment of sorafeni, the patients were divided into drug-resistant and sensitive types according to the imaging findings combined with serological index (AFP). The liver and paracancerous tissues were taken in each group, and the iTRAQ experiment was carried out with other liver cirrhosis patients and healthy donor liver tissue as the reference control, and the difference of tissue protein expression between the drug resistant and the sensitive groups was compared. GO, KEGG and other bioinformatics analysis, found the characteristic marker spectrum and the biological pathway involved.2, established the sorafeni drug resistance model of liver cancer cells in vitro. In the model, the expression difference of the characteristic markers was further verified by Western blot technology, and.3 was verified by the drug resistance model, PPARy antagonist and agonist, and the CCK8 method was used. The proliferation of hepatoma cells under the external intervention of PPAR gamma signaling pathway. Cell flow cytometry was used to study the effect of cell cycle arrest and apoptosis on the proliferation inhibition of hepatoma cells, and to explore the potential anti sorafeni resistance value of PPAR gamma antagonist. Results: 1, the expression difference between the current sorafenib sensitive group and the drug resistant group was 1. by using iTRAQ technique. More than 5 times more protein than 222, and the expression difference reached more than 2 times of protein 57, of which there were 26 expression up-regulated proteins and 31 down-regulated proteins; 2, GO analysis showed that the differential proteins involved small molecular metabolic processes, metabolic processes, carboxylic acid metabolism and metabolic processes of oxyacid, and KEGG signaling pathway analysis also indicated that The difference protein was enriched in sugar metabolism, fatty acid metabolism and glycolysis pathway; 3, the identification of 2 times more than the protein in vitro drug resistance model found that the differences of ITGA6, FN1, ICAM1, ITPA, BAX, TXNDC17 and MSH2 were consistent with the results of the body, and could be used as the candidate proteins for the mechanism study; 4, based on the bioinformatics analysis, the discovery of PPARs In particular, the expression of PPAR gamma was significantly changed in the drug resistance model. 5, PPAR gamma antagonist T0070907 was found to enhance the sensitivity of liver cancer cells to sorafenib, and the other antagonist GW9662 failed to achieve the same effect; 6, the combination of antagonist T0070907 and GW9662 failed to step up the cell sensitivity to sorafenib; 7, liver cancer was fine. Conclusion: 1, by comparing the tissue protein expression profiles of sorafenib and sensitive patients, a number of differential proteins are found to be involved in biological processes such as fatty acid metabolism and signaling pathways. 2, PPAR gamma may be a key factor in the occurrence of Sola Fini resistance in liver cancer, and its antagonist T007 0907, it can enhance the sensitivity of liver cancer cells and has potential clinical application value.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.7

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