【作者】 蒋国平；

【导师】 郑树森；

【作者基本信息】 浙江大学 ， 外科学（普外）， 2016， 博士

【摘要】 背景:肝癌为我国高发恶性肿瘤之一,每年约有38.3万人死于肝癌,约占全球肝癌死亡总数的51%。目前肝癌治疗手段有限,疗效不理想,病死率极高,是困扰我国群众健康的重大问题。肝癌的常见治疗手段包括外科手术、肝动脉栓塞化疗术和消融术等,对于无法手术患者,化疗也是重要手段之一。近年来新型分子靶向治疗技术的发展为肝癌治疗带来了全新视野。靶向药物索拉非尼可以通过抑制酪氨酸激酶阻止肿瘤新生血管的生成,还能经Raf/MEK/ERK信号传导通路直接抑制肿瘤生长。临床试验表明索拉非尼在肝癌晚期患者中展现了良好的疗效和耐受性,但存在耐药现象,其耐药机制尚不明确,一般认为与肿瘤细胞、肿瘤基质和个体遗传耐药性有关。相关研究表明肝癌耐药相关分子的表达变化能指示患者疗效敏感性,因此通过探索潜在生物标志物,建立索拉非尼治疗疗效的分子生物标志物谱,将有利于针对性给药,实现精准医疗;此外对于关键分子的机制研究,将解决耐药发生,并推动新药研发。方法:1、外科(肝切除/肝移植)术后复发患者接受索拉非尼治疗,根据影像学表现结合血清学指标(AFP)将入组患者分为耐药型和敏感型。取各组肝癌及癌旁组织,同时以其他肝硬化患者及健康供肝组织作为基准对照进行iTRAQ实验,比较耐药组与敏感组之间组织蛋白表达差异,进而开展GO、KEGG等生物信息学分析,发现特征标志物谱和所涉及的生物学通路。2、建立肝癌细胞体外索拉非尼耐药模型,在模型中采用Western blot技术对特征标志物的表达差异进一步验证。3、利用耐药模型,结合PPARy拮抗剂及激动剂,采用CCK8法检测PPARγ信号通路外在干预下肝癌细胞增殖情况的变化,细胞流式技术研究细胞周期阻滞和凋亡对肝癌细胞增殖抑制的作用,探索PPARγ拮抗剂潜在的抗索拉非尼耐药价值。结果:1、利用iTRAQ技术,发现索拉非尼敏感组和耐药组表达差异达到1.5倍以上的蛋白有222个,而表达差异达到2倍以上的蛋白57个,其中表达上调蛋白有26个,表达下调蛋白有31个;2、GO分析表明差异蛋白多涉及小分子代谢过程、分解代谢过程、羧酸代谢过程及含氧酸的代谢过程,而KEGG的信号通路分析同样表明,差异蛋白富集在糖代谢、脂肪酸代谢和糖酵解通路;3、利用体外耐药模型对2倍以上差异蛋白的验证发现包括ITGA6、FN1、ICAM1、ITPA、BAX、TXNDC17和MSH2等蛋白差异与体内结果一致,可作为机制研究的候选蛋白;4、基于生物信息学分析结果,发现PPARs特别是PPARγ在耐药模型中表达发生显著变化;5、发现PPARγ的拮抗剂T0070907能够增强肝癌细胞对索拉非尼的敏感性,另一个拮抗剂GW9662未能达到同样效果;6、,.拮抗剂T0070907及GW9662的联用未能进一步增强细胞对索拉非尼的敏感性;7、肝癌细胞对索拉非尼敏感性的增强是细胞周期阻滞所致。结论:1、通过比较索拉非尼耐药和敏感患者组织蛋白表达谱,发现多个差异蛋白,涉及脂肪酸代谢等生物学过程及信号通路;2、PPARγ可能是肝癌索拉非尼耐药发生的关键因子,其拮抗剂T0070907可增强肝癌细胞的敏感性,具有潜在的临床应用价值。更多还原

【Abstract】 Background:Primary liver cancer is one of the high incidence of malignant tumors in China,with limited treatment,lack of therapeutic efficacy and high mortality,which remains a major threat to public health in our country.About 383 thousand people die each year from liver cancer,accounting for about 51%of the total global cancer deaths.Surgery,transcatheter hepatic arterial chemoembolization and radiofrequency ablation are common approaches to the management of HCC,and chemotherapy is offered as a non-surgical option for the inoperable ones.Recent advances in molecular targeted therapy has brought new insight into studies of HCC treatment.Sorafenib,a molecular target agent,inhibits tumor angiogenesis by suppressing tyrosine kinase activity and directly inhibits tumor growth via Raf/MEK/ERK signaling pathway.Despite the favorable efficacy and safety in patients with advanced hepatocellular carcinoma,sorafenib induces drug resistance clinically,which has not been clearly understood and is usually related to the tumor cells,tumor stroma and individual genetic resistance.There are studies suggesting that changed expression of resistance-related molecules may indicate therapeutic efficacy and sensitivity of HCC treatment.Therefore,exploratory investigation of biomarker candidates for sorafenib efficacy will facilitate individual medication and further implementation of precision medicine.Moreover,interpretation of molecular mechanism based on key molecules can help overcome drug resistance and promote new drug development.Methods:Patients with postoperative recurrence after liver resection or liver transplantation received continuous sorafenib therapy,and were divided into sorafenib-resistant and sorafenib-sensitive based on results from imaging as well as serum markers(AFP).HCC and adjacent liver tissues were collected and used for iTRAQ analysis,with tissues from liver cirrhosis patients and healthy donors used as control.Protein profiles of sorafenib-resistant and sorafenib-sensitive groups were compared to identify the differentially expressed proteins,which were then annotated by GO and KEGG enrichment analysis to find specific biomarkers and biological pathways.We induced sorafenib resistance in HCC cells and verified the expression of identified biomarker candidates using western blot in these cells.The sorafenib-resistant cells,which were then treated with PPARγ antagonist and agonist,and phenotype alteration was detected,including cell proliferation analysis using CCK-8 assay,cell cycle and apoptosis analysis using cytometry,to investigate the potentially inhibitory effect of PPARy antagonist on sorafenib resistance.Results:1.By using iTRAQ analysis,we found 222 proteins with>1.5-fold change,of which 57 showed>2-fold change in expression levels,including 26 proteins upregulated and 31 downregulated.2.BP analysis demonstrated that the differentially expressed proteins function mostly in small molecule metabolism,degradation,carboxylic acid metabolism and oxoacid metabolism,and KEGG pathway analysis found carbon metabolism,fatty acid metabolism and glycolysis pathways involving these proteins.3.For proteins with>2-fold change,the alteration of ITGA6、FN1、ICAM1、ITPA、BAX、TXNDC17 and MSH2 in vitro expression was found consistent with that of in vivo expression,which made them important candidates in mechanism investigation.4.Based on the bioinformatic data,we found that the expression of PPARs,especially the PPARy,significantly changed in sorafenib-resistant cells.5.PPARγ antagonist T0070907 enhanced the sensitivity of HCC cells to sorafenib,whereas GW9662,another PPARγ antagonist,had no apparent effect.6.The combination of antagonist T0070907 and GW9662 did not result in a synergistic effect on sorafenib sensitivity.7.The enhanced sensitivity to sorafenib might be attributed to the cell cycle arrest induced by PPARy antagonists.Conclusions:1.The comparison of protein profiles between tissues from sorafenib-resistant and sorafenib-sensitive patients revealed multiple differentially expressed proteins that are involved in various biological processes and signaling pathways.2.PPARγ may play a pivotal role in the development of sorafenib resistance in HCC,and its antagonist T0070907 can enhance the sorafenib sensitivity of HCC cells,which suggests a potential value in clinical practice.更多还原