臺灣博碩士論文加值系統

理論上，T細胞於胸腺發育時，隨機將不同 T細胞接受器基因片段組裝，經此 V(D)J 重組步驟，最後產生出 T細胞接受器 (TCR)。而 T細胞接受器中的互補決定區 3 (CDR3) 已知是屬於高變異區域。利用三份來源不同的胸腺檢體，互補決定區 3 的研究使我們從中獲取了許多寶貴資訊。本研究著重於兩個主題，首先，我們調查了人類胸腺中，T 細胞 α 鏈接收器在變異區域(Variable region)及接合區域 (Joining region)的重組情形，亦解構出這三份檢體中的核苷酸組成、胺基酸組成、一種新型態的混合型 Jα T細胞接受器 (hybrid Jα TCR)及公有 T細胞接受器(Public TCR) 。第二，利用實驗方法與電腦程式 (MATLAB)，進行人類胸腺 T 細胞 α 鏈接收器庫的估計。本研究的貢獻在於發現互補決定區 3 的特色、提供支持 Vα-Jα 重組基因座收縮理論的證據以及探索公有 T 細胞接受器之形成機制。除此之外，我們估計出胸腺 T細胞 α鏈接收器庫(thymic TCRα repertoire)大小約略在10^8到10^9。

In theory, TCR is generated during T cell development in the thymus by a random process of assembling different gene segments, known as V(D)J recombination. The complementarity determining region (CDR) 3 is highly variable region of TCR. The studies of CDR3 from three thymic samples provide invaluable information. We address two topics. First, we investigated the recombination of human TCR variable (V) and junction (J) regions of α chain. Furthermore, the nucleotide usages, amino acid usages, a novel type of hybrid Jα TCR, and public TCR of three individuals were deciphered. Second, the thymic TCRα repertoire was assessed by experimental AmpliCot method and computer simulation program (MATLAB). Our first contribution is to exhibit the features of TCRα CDR3 and provide evidence in support of locus contraction mechanism of Vα-Jα recombination. Our second contribution is to explore the generation mechanism of public TCR. Importantly, the size of thymic TCRα repertoire is estimated to be 10^8 to 10^9.

Contents

口試委員會審定書…………………………………………………………………………………I
誌謝…………………………………………………………………………………………………II
中文摘要……………………………………………………………………………………………III
Abstract……………………………………………………………………………………………IV
Contents……………………………………………………………………………………………V
Abbreviations………………………………………………………………………………………VII
List of figures………………………………………………………………………………………IX
List of tables………………………………………………………………………………………XI
Chapter 1 Introduction………………………………………………………………………………1
1.1 T-cell………………………………………………………………………………………1
1.2 T-cell development…………………………………………………………………………1
1.3 Thymic selection……………………………………………………………………………2
1.4 TCR…………………………………………………………………………………………2
1.5 TCR rearrangement…………………………………………………………………………3
1.6 CDR3………………………………………………………………………………………4
1.7 CDR3 in autoimmune disease………………………………………………………………5
1.8 CDR3 in TCR-pMHC interaction…………………………………………………………5
1.9 CDR3 in TCR diversity……………………………………………………………………6
1.10 Aim of the Thesis…………………………………………………………………………7
Chapter 2 Materials and methods……………………………………………………………………9
2.1 Thymocytes isolation………………………………………………………………………9
2.2 RNA extraction and cDNA synthesis………………………………………………………9
2.3 DNA sequencing…………………………………………………………………………10
2.4 Sorting and aligning of raw sequencing data………………………………………………10
2.5 Manipulation of TCR CDR3 sequence……………………………………………………11
2.6 Nucleotide addition and N-AA……………………………………………………………11
2.7 Diversity estimation………………………………………………………………………12
2.8 Quantitative PCR of Vα distribution………………………………………………………12
2.9 AmpliCot…………………………………………………………………………………13
2.10 Statistics analysis…………………………………………………………………………14
Chapter 3 Results……………………………………………………………………………………15
3.1 Hot spots of TCRα rearrangement………………………………………………………15
3.2 Features of TCRα CDR3…………………………………………………………………17
3.2.1 Unique sequence ratio…………………………………………………………………17
3.2.2 Hybrid-Jα TCR…………………………………………………………………………18
3.2.3 Length distribution of TCRα CDR3……………………………………………………19
3.2.4 Amino acid usage of TCRα CDR3………………………………………………………20
3.3 Public TCR………………………………………………………………………………21
3.3.1 Public TCRα profiles……………………………………………………………………21
3.3.2 N-addition in Public TCR………………………………………………………………22
3.3.3 Usage of N region amino acid (N-AA) …………………………………………………23
3.3.4 Comparison of public sequences………………………………………………………25
3.4 T-cell repertoire……………………………………………………………………………26
3.4.1 Relative quantification of three thymus samples………………………………………26
3.4.2 Computer simulation of TCR diversity…………………………………………………26
3.4.3 AmpliCot for TCR diversity……………………………………………………………27
Chapter 4 Discussion………………………………………………………………………………28
4.1 Characteristics of TCR CDR3……………………………………………………………28
4.2 Hybrid-Jα provided a new thought in TCR recombination………………………………31
4.3 Biased amino acid shed light on immune disease detecting………………………………33
4.4 Public TCR may be a marker for specific immune response……………………………36
4.5 The size of TCR repertoire………………………………………………………………40
Chapter 5 Conclusion………………………………………………………………………………42
Figures………………………………………………………………………………………………43
Tables………………………………………………………………………………………………86
References…………………………………………………………………………………………100
Appendices………………………………………………………………………………………110

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