白塞病（一种自身免疫性疾病）发病机制的膜和核自身抗原抗体的鉴定
发布时间：2021-09-18 11:38
白塞病（BD）是一种罕见的系统性血管炎疾病,几乎会影响到身体的各个器官,其特征为眼部、口腔和粘膜溃疡。在某些情况下,血管炎的并发症会导致失明或死亡。没有人知道BD的确切原因,但该病可被归类为自身免疫性疾病。这种疾病经常发生在古丝绸之路沿线国家,在中国,土耳其和日本尤为常见。发达国家的总体发病率很低,因此长期以来无法引起主流科研机构的关注。因此,这种疾病的研究进展相对缓慢。到目前为止,该病仍处于无临床试验状态,无治疗状态,对发病机制尚无基本了解。超过40%的中国患者需要5年以上才能明确诊断出该病。目前的临床诊断仍然依赖于医生的经验判断,因此对该病的自身抗体和自身抗原的相关研究仍然薄弱。自身抗体是许多自身免疫疾病的重要实验室诊断工具,然而,截至目前为止,BD疾病特异性自身抗体还无法成功应用于国内外临床实践。本课题致力于通过建立一套完整的系统生物学方法,结合传统的生物技术和现代蛋白质组学筛选出一些BD疾病潜在自身抗原。在这项研究中,我们使用中国汉族患者的血清鉴定了四种BD疾病潜在自身抗原。膜联蛋白A1（AnnexinA1）,hnRNPC1/C2,NUMA的膜突和羧基（C）-末端氨基酸。简而言...
【文章来源】：北京科技大学北京市 211工程院校 教育部直属院校
【文章页数】：132 页
【学位级别】：博士
【文章目录】：
Acknowledgement
摘要
Abstract
Abbreviation
1 Introduction
2 Literature Review
2.1 History and epidemiology
2.2 Clinical manifestations and diagnosis
2.3 Causes of Pathogenesis
2.3.1 Genes are involved in BD Pathology
2.3.2 Role of Infectious agents and BD pathogenesis
2.4 Research progress in screening of BD autoantigens
2.4.1 Role of modern Techniques in screening of BDautoantigens
2.5 Research Contents and Approaches
3 Material and methods
3.1 HUVE Cell Line and screening of BD autoantigens
3.1.1 Sub Cell Culture
3.2 Main Reagents and instruments
3.3 Preparation of main working solutions/reagents
3.4 Experimental methods
3.4.1 Isolation of primary endothelial cells from umbilical cord vein
3.4.2 Passage of primary endothelial cells
3.4.3 Primary cryopreservation of endothelial cells
3.4.4 Cells recovery
3.4.5 RNA Extraction
3.4.6 RT PCR
3.4.7 Primer Designing
3.4.8 Target gene PCR amplification (50μL reaction system)
3.4.9 Plasmid construction and amplification
3.4.10 Enzyme double digestion
3.4.11 Ligation of target gene with pET 28 a (+) plasmid
3.4.12 DNA Transformation
3.4.13 Verification of transformed product
3.4.14 Construction of expression strains
3.4.15 IPTG induced expression
3.4.16 Purification of the target protein
3.4.17 Dialysis and Concentration of Purified Proteins
3.4.18 CA method for protein concentration determination
3.5 Validation of purified protein
3.5.1 SDS-PAGE and Western blotting
3.6 Mass spectrometry
3.7 Procedure for Enzyme Linked Immunosorbant assay (ELISA)
3.7.1 ELISA data analysis
3.8 Immunoprecipitation
3.9 Indirect immunofluorescence assay
3.10 Immunohistochemistry
3.11 Statistical analysis
4 Screening of autoantibodies (Annexin A1 and A2) in Behcet's Disease patientsby using bioinformatics tools
4.1 Cell culture
4.2 Sequence alignment and antigenic epitope prediction by usingbioinformatics tools
4.3 Cloning, expression and purification of AnnexinA1
4.4 Identification of Annexin A1 as autoantigen
4.5 In house development of ELISA
5 Identification of hnRNP C1/C2 as an autoantigen in patients with Behcet'sDisease
5.1 Structure, properties and biological functions of hnRNPs
5.2 Nuclear heterogeneous ribonucleoprotein and disease
5.3 Research progress on Behcet,s disease autoantigen
5.4 Expression and purification of hnRNP C1 C2 protein
5.5 Western Blotting
5.6 ELISA results
5.7 Statistical analysis
5.8 Discussion
6 Moesin expressions correlates its involvement in patients with Behcet's Disease
6.1 Subjects
6.2 Moesin cloning, expression and purification
6.3 Western Blotting
6.4 Verification of cross reactivity of ERM antibodies
6.5 Immunoprecipitation
6.6 ELISA
6.7 Clinical significance of BD patients
6.8 Resazurin assay experiments
6.9 Discussion
7 Circulation autoantibodies against C-terminus of NuMA in patients withBehcet's disease
7.1 Collection of samples
7.2 Indirect immunofluorescence assay
7.3 Plasmid Construction, expression and purification of NuMA C-terminalpeptide
7.4 The prevalence of antibody against C-NuMA
7.5 The clinical characteristics in BD patients
7.6 Discussion
8 Application of high throughput techniques in screening of Behcet's Disease autoantigens
8.1 Cell Chip Technology
8.1.1 Experimental results
8.2 Application of phage display technology in screening of BD autoantigens
8.2.1 Experimental Results and Discussion
8.3 Application of Mass spectrometry in screening of BD autoantigens
8.3.1 Identification of antigenic proteins
8.4 Experimental Materials
8.4.1 Establishment of experimental methods
8.5 Selection of serum for experimental use
8.5.1 Experimental steps
8.5.2 Results of experimental methods
8.6 Selection of experimental serum
8.7 Autoantigens detection with autoantibodies (Immunoassays)
8.8 Verification of autoantigens
8.9 Summary
9 Conclusion
References
作者简历及在学研究成果
学位论文数据集
【参考文献】：
期刊论文
[1]Endoscopic findings of gastrointestinal involvement in Chinese patients with Behcet’s disease[J]. Jun Zou,Yan Shen,Da-Nian Ji,Song-Bai Zheng,Jian-Long Guan. World Journal of Gastroenterology. 2014(45)
[2]膜联蛋白Ⅰ的结构和生物学功能[J]. 郭敦明,谈文峰,王芳. 医学综述. 2009(12)
[3]生物质谱在蛋白质组学研究中的应用[J]. 黄凌云,赵和平,丁勤学,何大澄. 现代仪器. 2004(01)
[4]生物质谱技术应用及进展[J]. 应万涛,钱小红. 军事医学科学院院刊. 2000(02)
[5]蛋白质组与生物质谱技术[J]. 钱小红. 质谱学报. 1998(04)
本文编号：3400068
【文章来源】：北京科技大学北京市 211工程院校 教育部直属院校
【文章页数】：132 页
【学位级别】：博士
【文章目录】：
Acknowledgement
摘要
Abstract
Abbreviation
1 Introduction
2 Literature Review
2.1 History and epidemiology
2.2 Clinical manifestations and diagnosis
2.3 Causes of Pathogenesis
2.3.1 Genes are involved in BD Pathology
2.3.2 Role of Infectious agents and BD pathogenesis
2.4 Research progress in screening of BD autoantigens
2.4.1 Role of modern Techniques in screening of BDautoantigens
2.5 Research Contents and Approaches
3 Material and methods
3.1 HUVE Cell Line and screening of BD autoantigens
3.1.1 Sub Cell Culture
3.2 Main Reagents and instruments
3.3 Preparation of main working solutions/reagents
3.4 Experimental methods
3.4.1 Isolation of primary endothelial cells from umbilical cord vein
3.4.2 Passage of primary endothelial cells
3.4.3 Primary cryopreservation of endothelial cells
3.4.4 Cells recovery
3.4.5 RNA Extraction
3.4.6 RT PCR
3.4.7 Primer Designing
3.4.8 Target gene PCR amplification (50μL reaction system)
3.4.9 Plasmid construction and amplification
3.4.10 Enzyme double digestion
3.4.11 Ligation of target gene with pET 28 a (+) plasmid
3.4.12 DNA Transformation
3.4.13 Verification of transformed product
3.4.14 Construction of expression strains
3.4.15 IPTG induced expression
3.4.16 Purification of the target protein
3.4.17 Dialysis and Concentration of Purified Proteins
3.4.18 CA method for protein concentration determination
3.5 Validation of purified protein
3.5.1 SDS-PAGE and Western blotting
3.6 Mass spectrometry
3.7 Procedure for Enzyme Linked Immunosorbant assay (ELISA)
3.7.1 ELISA data analysis
3.8 Immunoprecipitation
3.9 Indirect immunofluorescence assay
3.10 Immunohistochemistry
3.11 Statistical analysis
4 Screening of autoantibodies (Annexin A1 and A2) in Behcet's Disease patientsby using bioinformatics tools
4.1 Cell culture
4.2 Sequence alignment and antigenic epitope prediction by usingbioinformatics tools
4.3 Cloning, expression and purification of AnnexinA1
4.4 Identification of Annexin A1 as autoantigen
4.5 In house development of ELISA
5 Identification of hnRNP C1/C2 as an autoantigen in patients with Behcet'sDisease
5.1 Structure, properties and biological functions of hnRNPs
5.2 Nuclear heterogeneous ribonucleoprotein and disease
5.3 Research progress on Behcet,s disease autoantigen
5.4 Expression and purification of hnRNP C1 C2 protein
5.5 Western Blotting
5.6 ELISA results
5.7 Statistical analysis
5.8 Discussion
6 Moesin expressions correlates its involvement in patients with Behcet's Disease
6.1 Subjects
6.2 Moesin cloning, expression and purification
6.3 Western Blotting
6.4 Verification of cross reactivity of ERM antibodies
6.5 Immunoprecipitation
6.6 ELISA
6.7 Clinical significance of BD patients
6.8 Resazurin assay experiments
6.9 Discussion
7 Circulation autoantibodies against C-terminus of NuMA in patients withBehcet's disease
7.1 Collection of samples
7.2 Indirect immunofluorescence assay
7.3 Plasmid Construction, expression and purification of NuMA C-terminalpeptide
7.4 The prevalence of antibody against C-NuMA
7.5 The clinical characteristics in BD patients
7.6 Discussion
8 Application of high throughput techniques in screening of Behcet's Disease autoantigens
8.1 Cell Chip Technology
8.1.1 Experimental results
8.2 Application of phage display technology in screening of BD autoantigens
8.2.1 Experimental Results and Discussion
8.3 Application of Mass spectrometry in screening of BD autoantigens
8.3.1 Identification of antigenic proteins
8.4 Experimental Materials
8.4.1 Establishment of experimental methods
8.5 Selection of serum for experimental use
8.5.1 Experimental steps
8.5.2 Results of experimental methods
8.6 Selection of experimental serum
8.7 Autoantigens detection with autoantibodies (Immunoassays)
8.8 Verification of autoantigens
8.9 Summary
9 Conclusion
References
作者简历及在学研究成果
学位论文数据集
【参考文献】：
期刊论文
[1]Endoscopic findings of gastrointestinal involvement in Chinese patients with Behcet’s disease[J]. Jun Zou,Yan Shen,Da-Nian Ji,Song-Bai Zheng,Jian-Long Guan. World Journal of Gastroenterology. 2014(45)
[2]膜联蛋白Ⅰ的结构和生物学功能[J]. 郭敦明,谈文峰,王芳. 医学综述. 2009(12)
[3]生物质谱在蛋白质组学研究中的应用[J]. 黄凌云,赵和平,丁勤学,何大澄. 现代仪器. 2004(01)
[4]生物质谱技术应用及进展[J]. 应万涛,钱小红. 军事医学科学院院刊. 2000(02)
[5]蛋白质组与生物质谱技术[J]. 钱小红. 质谱学报. 1998(04)
本文编号：3400068
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