环形泰勒虫转化对B细胞细胞因子的产生及DC细胞抗原吞噬递呈能力的影响
发布时间：2021-11-06 21:04
环形泰勒虫是一种能够转化单核细胞/巨噬细胞、树突状细胞和B细胞的胞内寄生原虫。转化后的细胞能够无限制的增殖,其功能也因环形泰勒虫感染而改变。本研究将对环形泰勒虫转化细胞后,细胞表型及其功能变化开展研究工作,包括细胞表面标志分子、细胞因子、吞噬功能及抗原递呈效率等。在研究过程中,表面标志分子及细胞吞噬抗原的分析通过细胞流式技术实现;通过RT-PCR检测技术,对相关基因（包括细胞因子、吞噬相关蛋白和GTPase RAB家族蛋白）的转录水平进行检测分析。通过本研究,成功建立了环形泰勒虫转化的B淋巴细胞系。对转化后细胞的细胞因子（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）的转录水平和表面标志分子的表达水平进行了比对分析。通过流式细胞技术分析比对了正常B细胞和环形泰勒虫转化B细胞表面标志分子CD21、IgM和CD19（WC4）的表达情况。通过分析,99%的转化B细胞表达CD21,在第10代和20代的细胞中分别有14.26.5...
【文章来源】：中国农业科学院北京市
【文章页数】：100 页
【学位级别】：博士
【文章目录】：
摘要
abstract
英文缩略表
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
致谢(ACKNOWLEDGEMENT)
作者简历(RESUME)
本文编号：3480522
【文章来源】：中国农业科学院北京市
【文章页数】：100 页
【学位级别】：博士
【文章目录】：
摘要
abstract
英文缩略表
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
致谢(ACKNOWLEDGEMENT)
作者简历(RESUME)
本文编号：3480522
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