监测人TCRαβT细胞CDR3谱系漂移、TCR基因重排方法的建立和初步应用

发布时间：2018-04-01 14:09

  本文选题：双特异性抗体(BsAb)　切入点：基因扫描(GeneScan)　出处：《第一军医大学》2006年博士论文

【摘要】：背景：近年来，病毒感染、肿瘤、自身免疫病、移植等病理状态下T细胞的应答与耐受取得较大发展，对这些疾病状态下特异T细胞产生机制、监测方法、在个性化治疗中的应用、相应抗原表位疫苗的开发研究有着重大理论和潜在临床应用价值。目前，“ELISpot技术”、“FCM检测细胞内细胞因子”、“四聚体技术”等可检测密切相关(已知抗原)特异T细胞的功能和频数，但是不能提供特异T细胞的分子特征和整个样本中T细胞的组成信息。因每个T细胞有着自己独特的TCR CDR3分子，不同T细胞克隆具有不同序列的CDR3基因组成(不同长度)，从而形成多样性的CDR3基因谱型，CDR3区的序列决定其结构，从而决定TCR的特异性，或者说，CDR3相当于T细胞的“指纹”，监测CDR3序列的多态性和长度分布以及不同病理状态下的频率变化(谱系漂移)，可以反映特定T细胞克隆扩增的程度，从而反映T细胞功能状态。经典的克隆选择理论认为这些特异T细胞来源于T细胞受体库中已有的T细胞“优势增生”，但随着外周B细胞BCR“二次重排”的研究进展，，已证实外周T细胞同样存在TCR的“二次重排(编辑／修正)，即T细胞能以一种部分或全新的V(D)J重排的新受体取代原有的受体，从而能清除自身反应性细胞，或者是改变针对自身抗原的反应性，亦或更好的作用于微生物。对不同病理状态下特异T细胞TCR分子特征的鉴定和动态变化监测，探讨其和TCR“二次重排”的相关性，建立相应的研究技术和模型受到广泛的关注。 目的 (1)建立监测人TCR αβ T细胞CDR3谱系漂移的免疫扫描谱型分析技术(immunoscope spectratyping technique)，分析正常人外周血单个核细胞(peripheral blood mononuclear cell，PBMC)中TCR αβ T细胞CDR3谱系的多态性和长度分布；监测活动性慢性乙型肝炎(Chronic Hepatitis B，CHB)、急性T淋巴细胞白血病(T-lineage acute lymphoblastic leukemia，T-ALL)、外周造血干细胞(peripheral blood stem cells，PBSC)移植前后患者PBMC中TCR αβ T细胞CDR3谱系漂移情况，并鉴定T细胞株Jurkat的TCR分子特征。为感染、肿瘤、移植等疾病的细胞免疫应答机制、诊断、治疗研究提供新的思路与方法；为TCR二次重排(second rearrangement)研究提供监测TCR动态变化的技术； (2)建立监测人TCR αβ T细胞TCR基因重排的连接介导PCR方法(Ligation-
[Abstract]:Background: in recent years, the response and tolerance of T cells to viral infection, tumor, autoimmune disease, transplantation and other pathological conditions have been greatly developed.The development and research of the corresponding antigen epitope vaccine have great theoretical and potential clinical application value in individualized therapy.At present, "ELISpot technology", "FCM detection of intracellular cytokines", "tetramer technology" and so on can detect the function and frequency of specific T cells closely related (known antigens).However, the molecular characteristics of specific T cells and the composition of T cells in the whole sample can not be provided.Because each T cell has its own unique TCR CDR3 molecule, different T cell clones have different sequences of CDR3 gene composition (different lengths, thus forming a diversity of CDR3 gene type of CDR3 region sequence to determine its structure, thus determining the specificity of TCR.In other words, CDR3 is equivalent to a "fingerprint" of T cells, monitoring the polymorphism and length distribution of CDR3 sequences and frequency changes under different pathological conditions (lineage drift, which can reflect the extent of cloning and amplification of specific T cells.Thus, the function of T cells is reflected.The classical clone selection theory suggests that these specific T cells are derived from the "dominant proliferation" of T cells in the T cell receptor library, but with the development of the "secondary rearrangement" of BCR in peripheral B cells,It has been confirmed that peripheral T cells also have a "secondary rearrangement" of TCR (editing / rearrangement), in which T cells can replace the original receptors with a partial or new type of V(D)J rearrangement, thereby eliminating self-reactive cells.Either by changing the responsiveness to their own antigens, or by acting better on microbes.The identification and dynamic monitoring of the molecular characteristics of specific T cell TCR under different pathological conditions, the relationship between the molecular characteristics and TCR rearrangement, and the establishment of corresponding research techniques and models have been paid more and more attention.Purpose1) to establish an immunoscope analysis technique for monitoring the CDR3 lineage drift of human TCR 伪 尾 T cells.Analysis of peripheral blood mononuclear cells (PBMC) in normal subjects with peripheral blood mononuclearThe polymorphism and length distribution of CDR3 lineage of TCR 伪 尾 T cells in PBMCs, and the monitoring of chronic Hepatitis acute lymphoblastic in active chronic hepatitis B, acute T lymphoblastic leukemiaThe CDR3 lineage shift of TCR 伪 尾 T cells in PBMC patients before and after leukemia- T-ALL transplantation, and the TCR molecular characteristics of T cell line Jurkat were identified.To provide new ideas and methods for the study of cellular immune response mechanism, diagnosis and treatment of infection, tumor, transplantation and other diseases, and to provide a technique for monitoring the dynamic changes of TCR in the study of secondary rearrangement of TCR.Establishment of a ligation mediated PCR method for monitoring TCR gene rearrangement in human TCR 伪 尾 T cells
【学位授予单位】：第一军医大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R392


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