本文選題：雙特異性抗體(BsAb)　切入點(diǎn)：基因掃描(GeneScan)　出處：《第一軍醫(yī)大學(xué)》2006年博士論文

【摘要】：背景：近年來，病毒感染、腫瘤、自身免疫病、移植等病理狀態(tài)下T細(xì)胞的應(yīng)答與耐受取得較大發(fā)展，對(duì)這些疾病狀態(tài)下特異T細(xì)胞產(chǎn)生機(jī)制、監(jiān)測(cè)方法、在個(gè)性化治療中的應(yīng)用、相應(yīng)抗原表位疫苗的開發(fā)研究有著重大理論和潛在臨床應(yīng)用價(jià)值。目前，“ELISpot技術(shù)”、“FCM檢測(cè)細(xì)胞內(nèi)細(xì)胞因子”、“四聚體技術(shù)”等可檢測(cè)密切相關(guān)(已知抗原)特異T細(xì)胞的功能和頻數(shù)，但是不能提供特異T細(xì)胞的分子特征和整個(gè)樣本中T細(xì)胞的組成信息。因每個(gè)T細(xì)胞有著自己獨(dú)特的TCR CDR3分子，不同T細(xì)胞克隆具有不同序列的CDR3基因組成(不同長(zhǎng)度)，從而形成多樣性的CDR3基因譜型，CDR3區(qū)的序列決定其結(jié)構(gòu)，從而決定TCR的特異性，或者說，CDR3相當(dāng)于T細(xì)胞的“指紋”，監(jiān)測(cè)CDR3序列的多態(tài)性和長(zhǎng)度分布以及不同病理狀態(tài)下的頻率變化(譜系漂移)，可以反映特定T細(xì)胞克隆擴(kuò)增的程度，從而反映T細(xì)胞功能狀態(tài)。經(jīng)典的克隆選擇理論認(rèn)為這些特異T細(xì)胞來源于T細(xì)胞受體庫中已有的T細(xì)胞“優(yōu)勢(shì)增生”，但隨著外周B細(xì)胞BCR“二次重排”的研究進(jìn)展，，已證實(shí)外周T細(xì)胞同樣存在TCR的“二次重排(編輯／修正)，即T細(xì)胞能以一種部分或全新的V(D)J重排的新受體取代原有的受體，從而能清除自身反應(yīng)性細(xì)胞，或者是改變針對(duì)自身抗原的反應(yīng)性，亦或更好的作用于微生物。對(duì)不同病理狀態(tài)下特異T細(xì)胞TCR分子特征的鑒定和動(dòng)態(tài)變化監(jiān)測(cè)，探討其和TCR“二次重排”的相關(guān)性，建立相應(yīng)的研究技術(shù)和模型受到廣泛的關(guān)注。 目的 (1)建立監(jiān)測(cè)人TCR αβ T細(xì)胞CDR3譜系漂移的免疫掃描譜型分析技術(shù)(immunoscope spectratyping technique)，分析正常人外周血單個(gè)核細(xì)胞(peripheral blood mononuclear cell，PBMC)中TCR αβ T細(xì)胞CDR3譜系的多態(tài)性和長(zhǎng)度分布；監(jiān)測(cè)活動(dòng)性慢性乙型肝炎(Chronic Hepatitis B，CHB)、急性T淋巴細(xì)胞白血病(T-lineage acute lymphoblastic leukemia，T-ALL)、外周造血干細(xì)胞(peripheral blood stem cells，PBSC)移植前后患者PBMC中TCR αβ T細(xì)胞CDR3譜系漂移情況，并鑒定T細(xì)胞株Jurkat的TCR分子特征。為感染、腫瘤、移植等疾病的細(xì)胞免疫應(yīng)答機(jī)制、診斷、治療研究提供新的思路與方法；為TCR二次重排(second rearrangement)研究提供監(jiān)測(cè)TCR動(dòng)態(tài)變化的技術(shù)； (2)建立監(jiān)測(cè)人TCR αβ T細(xì)胞TCR基因重排的連接介導(dǎo)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 偽 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 偽 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 偽 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 偽 尾 T cells
【學(xué)位授予單位】：第一軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類號(hào)】：R392


本文編號(hào)：1695898