【作者】 高峰；

【导师】 郑树森；

【作者基本信息】 浙江大学 ， 外科学， 2009， 博士

【摘要】 前言:肝癌(HCC)是世界上第五位发病的恶性肿瘤,每年新发的肝癌诊断超过1,000,000例。早期检测是降低肝癌死亡率的关键步骤之一。在这些肝癌患者中,将近40%表现为AFP阴性。对于AFP阴性肝癌患者来说,了解肝癌分子机制和发现新的肝癌相关的生物标志物是迫切需要的。实验过程:本研究中,我们采用1D SDS-PAGE联合LC-ESI-MS/MS的非标记shotgun策略对AFP阴性HCC患者的肝癌和癌旁组织进行组织蛋白组学的分析;对癌和癌旁组织培养上清的分泌组学进行分析;对肝癌和健康对照的血浆进行血浆蛋白组学的分析。通过全方位的分析,全面了解AFP阴性肝癌的分子机制,并提供潜在的生物标记物数据源。对于组织蛋白组学,我们通过1D SDS-PAGE联合nano-UPLC ESI MS/MS(Q-TOF primier),对9例AFP阴性肝癌患者的癌和癌旁组织进行配对的组织蛋白组学研究,这个试验中使用的是PLGS 2.3搜索引擎。对于组织分泌组学,我们通过癌和癌旁组织无血清培养,再应用1 DSDS-PAGE联合MDLC ESI MS/MS(LTQ-Orbitrap)对4例AFP阴性肝癌患者的癌和癌旁组织的培养上清的分泌组学进行配对的组织分泌组学研究,这个试验中使用SEQUEST搜索在擎。对于血浆蛋白组学,我们通过免疫吸附进行丰度蛋白的分离,再应用1DSDS-PAGE联合nano-UPLC ESI MS/MS(Q-TOF primier)对3例AFP阴性肝癌患者及3例正常健康对照的血浆进行血浆蛋白组学研究。这个试验中使用X!TANDEM搜索在擎。结果:在组织蛋白组学部分,我们共鉴定到3908个蛋白,通过与癌旁比较,其中282个蛋白至少2倍上调表达,127个蛋白至少2倍下调表达。这种差异通过western验证是可信的。在组织分泌组学部分,我们共鉴定到了1302个蛋白,通过与癌旁比较,其中176个在癌组织的培养上清中至少2倍上调表达,200个在癌组织的培养上清中至少2倍下调表达。这些差异通过western验证是可信的。在血浆蛋白组学部分,我们共鉴定到了2779个蛋白,通过与正常人比较,其中107个蛋白在癌症患者血浆中高表达,102个表现为低表达。结论:我们第一次同时对AFP阴性肝癌患者进行组织蛋白组学、组织分泌蛋白组学和血浆蛋白组学研究,我们建立了一个完整的AFP阴性肝癌患者的蛋白组学数据库,通过不同的策略进行后期的功能和应用方面研究。更多还原

【Abstract】 Introduction:Hepatocellular carcinoma(HCC) ranks fifth in cancer frequency in the world with an estimated 1 million new cases occurring per year.Early detection is one of the key strategies in reducing liver cancer death.40%of these HCC cases show serum AFP negative.It is urgent need great to acknowledge the molecular mechanism and identify additional novel HCC markers for early detection of liver cancer in the AFP-negative cases.Experimental procedure:In this study,focus on the AFP negative cases,we utilize 1 D SDS-PAGE coupling with LC ESI-MS/MS label free shotgun strategy to analyze the tissue proteome of the cancerous and adjacent normal tissues,the tissue secretome of the cancerous and adjacent normal tissues,the plasma proteome of the AFP negative HCC cases and health controls.With the full proteomics research,we can acknowledge the molecular mechanism and reveal the potential biomarker candidates in the AFP negative HCC cases.To the tissue proteome,we utilize 1 D SDS-PAGE coupling with nanoUPLC ESI MS/MS((Q-TOF primier),perform the proteome research of 9 paired cancerous and their adjacent normal tissues.The search engine in this study is PLGS 2.3.To the tissue secretome,we culture the tissues in serum free media,then utilize 1 D SDS-PAGE coupling with MDLC ESI MS/MS(LTQ-Orbitrap),perform the secretome research of 4 paired cancerous and their adjacent normal tissues.The search engine in this study is SEQUEST.To the plasma proteome,we perform abundant protein fraction using immnoaffinity method,then utilize 1 D SDS-PAGE coupling with nanoUPLC ESI MS/MS((Q-TOF primier ),perform the plasma proteomics research of 3 paired plasma of the AFP negative HCC cases and health controls.The search engine in this study is X! TANDEM.Result:In the tissue proteome,we totally identified 3908 proteins,compared to the paired adjacent normal tissue,in which 282 proteins over-expressed at least 2 folds,and 127 proteins down-expressed at least 2 folds.The differences were validated by western blot.In the tissue secretome,we totally identified 1302 proteins,compared to the paired adjacent normal tissue,in which 176 proteins over-expressed at least 2 folds,and 200 proteins down-expressed at least 2 folds.The differences were validated by western blot.In the plasma proteome,we totally identified 2779 proteins,compared to the paired health controls,in which 107 proteins over-expressed at least 2 folds,and 102 proteins down-expressed at least 2 folds.Conclusion:For us,this is the first time to research the tissue proteome,tissue secretome and plasma proteome simultaneously,we have found a complete proteome database for AFP negative HCC cases,which enable us perform the further functional and applicable research.更多还原