TCR-CDR3片段ASSLSGENTEAF在肝脏移植急性免疫排斥过程中的调控作用与机制研究

Liver transplantation is currently the only effective mean for the treatment of liver failure. However, the change of T cell lineage drift was still unclear in the process of liver allograft rejection and immune tolerance. T cells are likely to have important roles in the process of liver allograft rejection. Analysis of the immune repertoire of T cell receptor (TCR), especially the CDR3 region is important for the determination of potential oligoclonal receptor skewing in a rejection population. In the preliminary studies we have found that there existed a TCR-CDR3 sequence by Arm-PCR and high throughput sequencing only in allograft rejection patients but not in stable patients and healthy volunteers. On the basis of the result we deduced that T cells expressing this clone might play an important role in the immune rejection. The project intends to predict the effects and mechanism of the specific TCR - CDR3 fragment ASSLSGENTEAF.. First, we intend to further expand the sample size and perform a dynamic monitoring in the process of rejection. Second, using mixed lymphocyte reaction model, investigate the effect of specific TCR - CDR3 by blocking it by specific antibody. Herein, we aimed at establishing and analyzing the TCR repertoire in liver allograft patients by means of multiplex PCR and CDR3-spectratyping with NGS to demonstrate any obvious CDR3 clonal expansions in patients with allograft as compared with control donors, which may help to provide an insight into the pathogenesis, diagnosis and treatment of the disease.

肝脏移植是目前治疗肝脏衰竭的唯一有效手段，移植排斥的诊断与治疗是肝脏移植重要环节。肝脏移植排斥过程中T细胞发挥重要作用，T细胞受体（TCR）是T 细胞识别HLA/抗原肽复合物并与之结合从而发挥杀伤毒性的关键元件，对其高度可变区（CDR3）的研究能够让我们更加深入的认识移植排斥过程。本研究组前期应用arm-PCR 与高通量测序，首次发现在多数排斥个体中存在CDR3片段ASSLSGENTEAF，而在未发生免疫排斥和健康个体中没有检测到，因此表达此片段的T细胞克隆可能在免疫排斥反应中发挥重要作用。本研究进一步扩大样本量，在发生排斥的肝穿组织切片中验证表达ASSLSGENTEAF的T淋巴细胞特异性存在；采用体外混合淋巴细胞反应模型，研究封闭该TCR谱系后对淋巴细胞的激活情况影响及其作用机制，探讨是否能通过抑制排斥特异性TCR克隆而实现调控肝脏移植后发生的免疫排斥反应，为免疫排斥的治疗提供新的思路。

近些年来随着外科手术技术进步、免疫抑制类药物的应用，肝脏移植的数量稳步增长，术后的生存率和生存质量不断提升。但是肝脏移植排斥仍然是肝脏移植后的较为严重的并发症之一。及时的诊断和治疗肝脏移植排斥需要对肝脏移植后的免疫过程的认识进一步加强。本研究扩大了前期研究的样本量，明确了TCR-CDR3 片段 ASSLSGENTEAF在肝脏移植排斥患者不具有普遍意义，应用TCR-CDR3 片段 ASSLSGENTEAF抗体无法抑制免疫排斥的发生，并且目前筛选出的片段无法作为肝脏移植排斥的标志物，但是通过对肝脏移植排斥和稳定患者TRB-CDR3组库的分析，可以通过D50等五个指标的组合大致区分移植排斥、稳定和正常个体，下一步经过对疾病和携带病毒的具体分组分析，有望开发出疾病诊断的灵敏的标志物。本项目的顺利实施，为开发针对灵敏诊断肝脏移植排斥的的方法提供思路，为肝脏移植的免疫过程的研究提供了研究基础和新的科学理论依据和实验证据，具有重要的理论意义和应用价值。