環(huán)形泰勒蟲是一種能夠轉(zhuǎn)化單核細胞/巨噬細胞、樹突狀細胞和B細胞的胞內(nèi)寄生原蟲。轉(zhuǎn)化后的細胞能夠無限制的增殖,其功能也因環(huán)形泰勒蟲感染而改變。本研究將對環(huán)形泰勒蟲轉(zhuǎn)化細胞后,細胞表型及其功能變化開展研究工作,包括細胞表面標志分子、細胞因子、吞噬功能及抗原遞呈效率等。在研究過程中,表面標志分子及細胞吞噬抗原的分析通過細胞流式技術(shù)實現(xiàn);通過RT-PCR檢測技術(shù),對相關(guān)基因（包括細胞因子、吞噬相關(guān)蛋白和GTPase RAB家族蛋白）的轉(zhuǎn)錄水平進行檢測分析。通過本研究,成功建立了環(huán)形泰勒蟲轉(zhuǎn)化的B淋巴細胞系。對轉(zhuǎn)化后細胞的細胞因子（IL-1α,IL-1β,IL-4,IL-6,IL-8,IL-10,IL-16,TGF-β₁,TNF-α,LT-α/TNF-β,IFN-α和IFN-β）的轉(zhuǎn)錄水平和表面標志分子的表達水平進行了比對分析。通過流式細胞技術(shù)分析比對了正常B細胞和環(huán)形泰勒蟲轉(zhuǎn)化B細胞表面標志分子CD21、IgM和CD19（WC4）的表達情況。通過分析,⁹9%的轉(zhuǎn)化B細胞表達CD21,在第10代和20代的細胞中分別有14.2⁶.5... 

【文章來源】：中國農(nóng)業(yè)科學院北京市

【文章頁數(shù)】：100 頁

【學位級別】：博士

【文章目錄】：
摘要
abstract
英文縮略表
CHAPTER Ⅰ INTRODUCTION
    1.1 THEILERIA ANNULATA BIOLOGY
        1.1.1 Taxonomy and life cycle
        1.1.2 Epidemiological features of Theileria annulata
        1.1.3 Tropical theileriosis
    1.2 CHANGES IN THEILERIA ANNULATA-TRANSFORMED CELLS
        1.2.1 Parasite survival in transformed lymphocyte
        1.2.2 Host cell deformation
        1.2.3 Cell surface markers
        1.2.4 Impaired endocytosis rate
    1.3 HOST IMMUNE RESPONSE FOR THEILERIA ANNULATA
        1.3.1 Host first line of defense against Theileria annulata
        1.3.2 Pathological changes and cytokine production in T.annulata infection
    1.4 APPROACHES OF VACCINE DEVELOPMENT
        1.4.1 Live vaccine
        1.4.2 Subunit vaccine
    1.5 RATIONALE AND OBJECTIVES OF THE STUDY
CHAPTER Ⅱ ESTABLISHMENT AND EXPRESSION OF CYTOKINES FROM THEILERIA ANNULATA-INFECTED BOVINE B CELL LINE
    2.1 INTRODUCTION
    2.2 MATERIALS AND METHODS
        2.2.1 Reagents and antibodies
        2.2.2 Experimental animals
        2.2.3 Magnetic cell separation
        2.2.4 Cell line establishment and maintenance
        2.2.5 Cytotoxicity assay
        2.2.6 Antigenic stimulation
        2.2.7 Theileriacidal treatment
        2.2.8 Flow cytometry analysis
        2.2.9 PCR and sequencing analysis
        2.2.10 RNA extraction and cDNA synthesis
        2.2.11 Quantitative PCR analysis
        2.2.12 Statistical analysis
    2.3 RESULTS
        2.3.1 Confirmation of experimental animals for piroplasmosis
        2.3.2 Establishment of B cell line
        2.3.3 B cell line specificity and purity analysis
        2.3.4 Identity analysis of T.annulata strain
        2.3.5 Optimized concentration of antigen and Theileriacidal drug
        2.3.6 Optimization of cytokines primers
        2.3.7 Cytokines
    2.4 DISCUSSION
CHAPTER Ⅲ ENDOCYTOSIS MECHANISM AND EFFICACY IN NORMAL AND THEILERIA ANNULATA TRANSFORMED BOVINE DENDRITIC CELLS
    3.1 INTRODUCTION
    3.2 MATERIALS AND METHODS
        3.2.1 Reagents and antibodies
        3.2.2 Sampling
        3.2.3 Cell isolation and culture
        3.2.4 MoDC generation and TaDC cell line maintenance
        3.2.5 Antigen uptake
        3.2.6 Pathways of endocytosis
        3.2.7 Flow cytometric analysis
        3.2.8 Primers optimization
        3.2.9 RNA extraction and Real-Time qPCR
        3.2.10 Data analyses
    3.3 RESULTS
        3.3.1 TaDC cell line
        3.3.2 Endocytosis efficacy
        3.3.3 Clathrin-dependent endocytosis
        3.3.4 Clathrin-independent endocytosis
        3.3.5 Upgraded endocytosis
        3.3.6 Expression level of chemokine’s and mannose receptors
    3.4 DISCUSSION
CHAPTER Ⅳ INFLUENCE OF THEILERIA ANNULATA ON THE FUNCTIONS OF BOVINE ANTIGEN PRESENTING DENDRITIC CELLS FOR STIMULATION OF T LYMPHOCYTES PROLIFERATION
    4.1 INTRODUCTION
    4.2 MATERIALS AND METHODS
        4.2.1 Materials and reagents
        4.2.2 Sample collection and cell isolation
        4.2.3 Preparation of antigen presenting cells
        4.2.4 T lymphocytes proliferation assay
        4.2.5 Antigen mobilization assay
        4.2.6 Flow cytometry analysis
        4.2.7 Primers optimization
        4.2.8 RNA isolation and Real-Time qPCR
        4.2.9 Data analysis
    4.3 RESULTS
        4.3.1 Cells purity and MHC class molecules
        4.3.2 Rate of T lymphocytes proliferation
        4.3.3 Antigen transfer to lymphocytes
        4.3.4 Rab family genes primer optimization
        4.3.5 Rho small GTPase expression in TaDC cell line
    4.4 DISCUSSION
CHAPTER Ⅴ CONCLUSION
REFERENCES
APPENDICES
    APPENDICES 1.1 PROTOCOLS
        Appendices 1.1.1: Cells isolation
        Appendices 1.1.2: Antigen presentation for T lymphocytes proliferation
    APPENDICES 1.2 BUFFERS AND SOLUTIONS
        Appendices 1.2.1: Complete cell culture medium
        Appendices 1.2.2: 1×PBS buffer composition
        Appendices 1.2.3: MACS buffer composition
        Appendices 1.2.4: Cell culture preservation medium
致謝(ACKNOWLEDGEMENT)
作者簡歷(RESUME)



本文編號：3480522