白塞�。˙D）是一種罕見的系統(tǒng)性血管炎疾病,幾乎會影響到身體的各個器官,其特征為眼部、口腔和粘膜潰瘍。在某些情況下,血管炎的并發(fā)癥會導(dǎo)致失明或死亡。沒有人知道BD的確切原因,但該病可被歸類為自身免疫性疾病。這種疾病經(jīng)常發(fā)生在古絲綢之路沿線國家,在中國,土耳其和日本尤為常見。發(fā)達(dá)國家的總體發(fā)病率很低,因此長期以來無法引起主流科研機(jī)構(gòu)的關(guān)注。因此,這種疾病的研究進(jìn)展相對緩慢。到目前為止,該病仍處于無臨床試驗狀態(tài),無治療狀態(tài),對發(fā)病機(jī)制尚無基本了解。超過40%的中國患者需要5年以上才能明確診斷出該病。目前的臨床診斷仍然依賴于醫(yī)生的經(jīng)驗判斷,因此對該病的自身抗體和自身抗原的相關(guān)研究仍然薄弱。自身抗體是許多自身免疫疾病的重要實驗室診斷工具,然而,截至目前為止,BD疾病特異性自身抗體還無法成功應(yīng)用于國內(nèi)外臨床實踐。本課題致力于通過建立一套完整的系統(tǒng)生物學(xué)方法,結(jié)合傳統(tǒng)的生物技術(shù)和現(xiàn)代蛋白質(zhì)組學(xué)篩選出一些BD疾病潛在自身抗原。在這項研究中,我們使用中國漢族患者的血清鑒定了四種BD疾病潛在自身抗原。膜聯(lián)蛋白A1（AnnexinA1）,hnRNPC1/C2,NUMA的膜突和羧基（C）-末端氨基酸。簡而言... 

【文章來源】：北京科技大學(xué)北京市 211工程院校 教育部直屬院校

【文章頁數(shù)】：132 頁

【學(xué)位級別】：博士

【文章目錄】：
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
作者簡歷及在學(xué)研究成果
學(xué)位論文數(shù)據(jù)集


【參考文獻(xiàn)】：
期刊論文
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