γδT细胞抗原识别机制研究：热休克蛋白的直接识别以及HBV-TCR CDR3δ多肽结合特性和结合多肽的筛选

发布时间：2018-03-23 00:39

本文选题：细胞　切入点：抗原　出处：《中国协和医科大学》2005年博士论文　论文类型：学位论文

【摘要】：γδT细胞兼具特异性和非特异性免疫应答双重特征，其在机体免疫应答中所处的进化地位和生物学意义与功能有待于进一步阐明。因此，揭示γδ抗原识别受体(TCR)抗原识别的规律，将有助于对上述理论问题进行澄清，并可为免疫学应用提供新的策略与手段。本论文旨在研究γδT细胞对热休克蛋白(HSP)的识别机制和探讨乙型肝炎病毒感染患者TCR Vδ 2的抗原识别机理，以及以TCR CDR3δ为探针筛选和鉴定γδ TCR识别的表位多肽。 (一)人类γδ T细胞对细胞表面热休克蛋白(membrane HSPs)识别机制的研究热休克蛋白在免疫应答中主要作用包括：1)与表位肽形成HSPs-peptide复合物，表达在细胞表面，参与抗原提呈过程；2)作为靶分子直接为免疫细胞所识别，诱导产生免疫应答。针对上述问题，本文首先重点研究了在γδT细胞识别HSP分子的过程中，HSP分子究竟是作为抗原提呈分子还是作为抗原这一基本问题，继而初步探讨了HSP分子和γδT细胞相互作用的生物学意义。为此，首先我们以EB病毒(EBV)转化B淋巴细胞(LCL细胞)作为细胞模型，探讨了细胞转化这一应激事件对细胞膜表面HSP分子表达水平的影响；其次应用siRNA干扰技术，分别敲除HSP60和HSP70分子，观察LCL细胞上述基因表达水平与γδT细胞对LCL细胞毒活性间的相关性；最后，利用体外增殖实验，探讨HSP分子是否对γδT细胞具有直接刺激作用。研究结果显示，在LCL细胞表面，HSP60和HSP70分子均可在转化应激的诱导下表达增高。HSP70分子表达下调后，，γδT细胞对自体和异体来源的LCL细胞毒活性均受到抑制，与对照组相比具有显著统计学意义(P＜0.05)，而HSP60分子表达水平对γδT细胞杀伤活性的影响较小。[~3H]-TdR掺入实验证实了HSP72分子可以直接刺激γδT细胞发生增殖。上述结果不仅在理论上支持了γδT细胞通过直接识别HSP分子参与早期免疫应答，尤其在对肿瘤的免疫监视，同时也为以HSP70分子(尤其是HSP72分子)为
[Abstract]:The evolutionary status, biological significance and function of 纬未 T cells in immune response need to be further elucidated. Therefore, the rules of recognition of 纬未 antigen recognition receptor TCRs are revealed. It will be helpful to clarify the above theoretical problems and provide new strategies and means for immunological application. The purpose of this study was to investigate the mechanism of 纬未 T cell recognition of heat shock protein (HSPs) and the antigen recognition mechanism of TCR V 未 2 in patients with hepatitis B virus infection, and to screen and identify the epitope peptides recognized by 纬未 TCR using TCR CDR3 未 as a probe. (1) study on the recognition mechanism of HSPs by human 纬未 T cells on cell surface heat shock protein membrane (HSPs). The main roles of heat shock protein in immune response include: 1) forming HSPs-peptide complex with epitope peptide, expressing on cell surface, participating in antigen presentation process, and acting as target molecule directly recognized by immune cells. In view of the above problems, this paper focuses on the basic question of whether the HSP molecule is the antigen presenting molecule or the antigen presenting molecule in the recognition of HSP molecule by 纬未 T cells. The biological significance of interaction between HSP molecules and 纬未 T cells was discussed. The effect of the stress event of cell transformation on the expression of HSP molecules on cell membrane surface was studied. Secondly, siRNA interference technique was used to knock out HSP60 and HSP70 molecules, respectively. To observe the correlation between the expression of these genes in LCL cells and the cytotoxicity of 纬未 T cells to LCL cells. Finally, to investigate whether HSP molecules can directly stimulate 纬未 T cells by in vitro proliferation test. On the surface of LCL cells, both HSP60 and HSP70 molecules were increased and down-regulated under the induction of transformation stress, and the cytotoxic activity of 纬未 T cells to autogenous and allogeneic LCL cells was inhibited. Compared with the control group, there was significant statistical significance (P < 0.05), while the expression of HSP60 had little effect on the cytotoxicity of 纬未 T cells. The results of [3H] -TdR incorporation showed that HSP72 could directly stimulate the proliferation of 纬未 T cells. It is suggested that 纬未 T cells take part in early immune response by directly recognizing HSP molecules. Especially in the immune surveillance of tumors, but also in the case of HSP70 molecules (especially HSP72 molecules).
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R392

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