细胞膜蛋白和血清外泌体的分离与癌症蛋白质组研究

发布时间：2021-11-15 09:39

　　细胞膜蛋白和细胞分泌的外泌体在生物系统中起着重要的作用。然而,由于自身的疏水性、动态差异以及膜脂的限制,膜蛋白的提取仍然非常具有挑战性,有必要发展新的分离策略,促进膜蛋白的功能研究,特别是在癌症研究方面。外泌体是信号传导的主要参与者,它们与癌症的发生、发展和转移密切相关,可用于癌症的诊断和治疗。定量比较细胞膜蛋白和外泌体蛋白在癌症组和对照组中的变化,将有助于发现癌症诊断生物标志物,探索癌症发生和转移的分子机制。本文的主要研究成果如下。首先,本文发展了基于碳纳米材料的膜蛋白和膜相关蛋白提取富集新方法。具有高比表面积的纳米材料具有疏水-疏水-脂质相互作用的能力,我们发现溶于裂解缓冲液中的石墨烯和氧化石墨烯能够提取细胞膜蛋白,并显著提高富集效率。液相色谱质谱分析表明,石墨烯（50-200 nm）和氧化石墨烯（50-200 nm）比膜蛋白提取试剂盒能富集更多种类的膜蛋白。将石墨烯进一步应用于正常细胞和癌细胞膜蛋白的富集,我们分别鉴定出1079和872种蛋白,其中56.5%和60.5%为膜蛋白。特别是在癌细胞中,有241个蛋白发生显著变化。我们采用qRT-PCR先验证了候选蛋白的基因表达,并对通... 

【文章来源】：上海交通大学上海市 211工程院校 985工程院校 教育部直属院校

【文章页数】：135 页

【学位级别】：博士

【文章目录】：
摘要
Abstract
Abbreviations
CHAPTER1:Introduction to membrane proteins and exosomes
    1.1 Proteomics and quantitative proteomics research methods
        1.1.1 Gel-based proteomic quantification:2-DE and 2D-DIGE
        1.1.2 Stable isotope labeling by amino acid in cell culture
        1.1.3 Isobaric tag for relative and absolute quantitation and tandem mass tags
        1.1.4 Label-free quantification
    1.2 Membrane proteins
        1.2.1 Importance of membrane proteins
        1.2.2 Challenges in membrane protein study
        1.2.3 Current methodologies of membrane protein extraction
            1.2.3.1 Precipitation and density gradient centrifugation
            1.2.3.2 Extraction of membrane proteins by cross-linking
            1.2.3.3 Membrane proteins extraction with cell-surface shaving
            1.2.3.4 Immunoaffinity techniques for enriching membrane proteins
            1.2.3.5 Ionic liquid for membrane protein extraction
            1.2.3.6 Nanomaterials based membrane proteins extraction method
    1.3 Exosomes
        1.3.1 Exosomes biogenesis
        1.3.2 Exosomes enrichment methods
        1.3.3 Exosome characterization methods
        1.3.4 Exosomes in cancer
        1.3.5 Exosomes as diagnostic tools
        1.3.6 Exosomes as therapeutic tools
    1.4 Cancer
    1.5 The background,purpose,and content of this research
        1.5.1 The background
        1.5.2 Research purposes
        1.5.3 Research content
Chapter2:Graphene and graphene oxide as a solid matrix for extraction of membrane and membrane-associated proteins
    2.1 Introduction
    2.2 Experimental materials,reagents,and instruments
    2.3 Experimental methods
        2.3.1 Normal and lung cancer cell culture
        2.3.2 Feasibility test for cellular membrane and nanomaterial interaction
        2.3.3 Membrane proteins extraction with Mem-PER~(TM) Kit
        2.3.4 Membrane proteins extraction with nanomaterials
        2.3.5 Membrane proteins separation
        2.3.6 Sample processing prior to mass spectrometry analysis
        2.3.7 Desalts of peptides
        2.3.8 Mass spectrometry based protein identification and label free quantification
        2.3.9 Cellular RNA extraction
        2.3.10 Real-time fluorescence quantitative PCR
        2.3.11 Western blotting
        2.3.12 Bio-informatics analysis
    2.4 Results and discussion
        2.4.1 Strategy of nanomaterials and kit membrane proteins extraction
        2.4.2 Membrane lipid-nanomaterial interaction analysis
        2.4.3 Comparisons of membrane protein extraction yields for nanomaterials selection
        2.4.4 Membrane proteins electrophoretic separation and analysis
        2.4.5 Membrane protein identification and analysis
        2.4.6 Evaluation and validation of developed method
            2.4.6.1 BEAS-2B and95D cell membrane protein extraction and separation
            2.4.6.2 Membrane and membrane-associated proteome analysis
            2.4.6.3 Lung cancer related membrane and membrane-associated proteins
            2.4.6.4 qRT-PCR verification of differentially expressed membrane proteins
            2.4.6.5 Candidate proteins selection for verification
            2.4.6.6 Immunoblot validation of candidate proteins
    2.5 Summary
Chapter3:Discovery of exosomal proteins from human serum and cell culture media for liver cirrhosis and liver cancer
    3.1 Introduction
    3.2 Experimental materials,reagents and instruments
    3.3 Experimental methods
        3.3.1 Human blood collection and processing
        3.3.2 Normal and liver cancer cell culture
        3.3.3 Exosomes isolation and enrichment from human serum
        3.3.4 Exosomes isolation and enrichment from culture media
        3.3.5 Exosomes morphological analysis by transmission electron microscopy
        3.3.6 Nanoparticles tracking analysis
        3.3.7 Western blot analysis
        3.3.8 Nano LC-MS/MS and label free quantification
    3.4 Results and discussion
        3.4.0 Strategy for exosomes preparation from human serum and culture media
        3.4.1 Exosomes enrichment and characterization
        3.4.2 Electrophoretic separation of exosomal protein
        3.4.3 Exosomal protein identification
        3.4.4 Cross-examination of identified exosomal proteins with existing database
        3.4.5 Comparative proteomic analysis of serum exosomes from liver cancer,liver cirrhosis,and healthy control
        3.4.6 Comparative proteomic analysis of cell derived exosomes
        3.4.7 Candidate exosomal proteins selection through combined data analysis
        3.4.8 Western blot verification of candidate proteins
        3.4.9 Summary
Chapter4:Conclusions,novelty,and outlook
References
Main instruments
List of differentially expressed membrane proteins
Acknowledgements
List of publications


【参考文献】：
期刊论文
[1]Cancer incidence and mortality in China, 2014[J]. Wanqing Chen,Kexin Sun,Rongshou Zheng,Hongmei Zeng,Siwei Zhang,Changfa Xia,Zhixun Yang,He Li,Xiaonong Zou,Jie He.  Chinese Journal of Cancer Research. 2018(01)



本文编号：3496525