細胞膜蛋白和細胞分泌的外泌體在生物系統(tǒng)中起著重要的作用。然而,由于自身的疏水性、動態(tài)差異以及膜脂的限制,膜蛋白的提取仍然非常具有挑戰(zhàn)性,有必要發(fā)展新的分離策略,促進膜蛋白的功能研究,特別是在癌癥研究方面。外泌體是信號傳導的主要參與者,它們與癌癥的發(fā)生、發(fā)展和轉移密切相關,可用于癌癥的診斷和治療。定量比較細胞膜蛋白和外泌體蛋白在癌癥組和對照組中的變化,將有助于發(fā)現癌癥診斷生物標志物,探索癌癥發(fā)生和轉移的分子機制。本文的主要研究成果如下。首先,本文發(fā)展了基于碳納米材料的膜蛋白和膜相關蛋白提取富集新方法。具有高比表面積的納米材料具有疏水-疏水-脂質相互作用的能力,我們發(fā)現溶于裂解緩沖液中的石墨烯和氧化石墨烯能夠提取細胞膜蛋白,并顯著提高富集效率。液相色譜質譜分析表明,石墨烯（50-200 nm）和氧化石墨烯（50-200 nm）比膜蛋白提取試劑盒能富集更多種類的膜蛋白。將石墨烯進一步應用于正常細胞和癌細胞膜蛋白的富集,我們分別鑒定出1079和872種蛋白,其中56.5%和60.5%為膜蛋白。特別是在癌細胞中,有241個蛋白發(fā)生顯著變化。我們采用qRT-PCR先驗證了候選蛋白的基因表達,并對通... 

【文章來源】：上海交通大學上海市 211工程院校 985工程院校 教育部直屬院校

【文章頁數】：135 頁

【學位級別】：博士

【文章目錄】：
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


【參考文獻】：
期刊論文
[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)



本文編號：3496525