结核病的gamma delta T细胞特异性诊断及其相关配体鉴定研究

Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis, which affects almost any tissues or organs of the body. With high prevalence, morbidity and mortality, tuberculosis has brought great challenges to humans. The short of efficient diagnostic methods has seriously affected the timely diagnosis of tuberculosis. γδT cells are implicated in the first line of the immune defense against infection and can be activated at the earliest stage of Mycobacterium tuberculosis infection. γδT cells play an important role in the removal of pathogens and the body's immune response against Mycobacterium tuberculosis infection. In this study, multiplex PCR and high-throughput sequencing will be performed for the analysis of the sequences of CDR3δ of γδT cells from healthy individuals and patients with tuberculosis in order to identify the specific characteristics of CDR3δ polymorphisms associated with Mycobacterium tuberculosis infection. Then we will identify the predominant sequences and the ligands recognized by γδT cells though an established method based on the binding specificity of CDR3δ in our laboratory. This study will establish an immunological method based on CDR3δ polymorphism of γδT cells for early diagnosis of TB and identify the ligands recognized by γδT cells, which lays the foundation for further elucidating the mechanism of γδT cells in the immune response against Mycobacterium tuberculosis infection.

结核病是一种由结核杆菌引起的全身多系统多器官的疾病，因其感染率，发病率和死亡率很高，给人类的健康带来了巨大的挑战。由于诊断方法所限，严重影响了结核病的及时诊断。γδT细胞处于抗感染免疫的防御一线，能在结核杆菌感染的最早期活化，清除病原体，在机体对抗结核杆菌感染的免疫应答发挥重要作用。本研究拟通过多重PCR和高通量测序的方法，对健康人和结核病患者的γδT细胞CDR3δ序列进行对比分析，找到特异性的结核杆菌感染相关的CDR3δ多态性特征，并选择其中的优势序列，应用实验室前期已建立一种基于CDR3δ肽的结合特异性的筛选方法，鉴定出γδT细胞所识别的相关配体。本研究能够建立一个基于γδT细胞CDR3δ多态性的结核病早期诊断的免疫学方法，鉴定出γδT细胞所识别的相关配体，为深入阐明γδT细胞在抗结核杆菌感染的免疫应答中的作用机制奠定基础。

兼具特异性和非特异性免疫应答双重特征的γδT 细胞在结核杆菌感染的早期就能迅速活化，启动抗感染免疫应答，对宿主产生保护作用。然而，其识别和启动抗结核杆菌免疫应答的作用机制尚不完全清楚。.本课题从结核杆菌感染相关γδT 细胞免疫组库特征入手，以感染相关的特异性的优势CDR3δ序列作为探针，筛选和鉴定γδT 细胞识别的结核杆菌相关表位/蛋白配体。研究发现结核病患者外周血γδT 细胞比例显著降低，分泌IFN-g的γδT 细胞也显著减少；结核病患者外周血γδT 细胞TCR CDR3免疫组库多样性减少，不同基因片段的取用频率发生了很大变化；用结核杆菌感染相关的CDR3δ优势序列筛选到4 条能与探针结合的十二肽表位和8 个结核杆菌蛋白；结核杆菌蛋白Rv0002 能有效刺激结核病患者外周血γδT 细胞的活化和增殖。.本课题首次获得了结核杆菌感染特异性的CDR3δ谱系变化特征，为结核病诊断和转化医学研究奠定基础；首次通过结核杆菌蛋白芯片技术筛选到了γδT 细胞识别的结核杆菌蛋白Rv0002，为γδT 细胞抗结核杆菌感染的机制研究提供了分子基础。