原发性胆汁性肝硬化患者B细胞受体CDR3免疫组库特征的初步研究

Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by portal inflammation and immune-mediated destruction of intrahepatic bile ducts, which often leads to cirrhosis and liver failure. B cells are likely to have important roles in the pathogenesis of PBC. Analysis of the immune repertoire of B cell receptor (BCR), especially the CDR3 region is important for the determination of potential oligoclonal receptor skewing in an autoantibody population. Previous attempts to investigate the role of B cell clones in PBC by screening the BCR repertoires were hampered by the sheer size and complexity of the repertoires. The ability to perform such analysis has been recently facilitated by the development of “next-generation sequencing technology (NGS)” and multiplex PCR for amplifying V(D)J recombination for BCR, directly from whole blood. Millions of BCR sequences can be read in parallel from a single sample. The dynamics of an adaptive immune response, which is based on clonal expansion and contraction, can be monitored in real time at high sensitivity and the global properties of the adaptive immune repertoires can be studied. Herein, we aimed at establishing and analyzing the BCR repertoire in PBC patients by means of multiplex PCR and CDR3-spectratyping with NGS to demonstrate any obvious CDR3 clonal expansions in patients with PBC as compared with control donors, which may help to provide an insight into the pathogenesis, diagnosis and treatment of the disease.

原发性胆汁性肝硬化（PBC）是一种原因不明的自身免疫性肝脏疾病，免疫学特点是血清中出现抗线粒体抗体（AMAs）及自身免疫介导的肝内中小胆管损伤。B细胞参与PBC免疫损伤机制，其抗原受体BCR重链CDR3区最具多样性，赋予机体识别各种抗原、产生特异性体液免疫应答的巨大潜能，构成容量巨大的免疫组库。早期由于受研究技术所限，人们对PBC患者BCR多样性的认识并不全面，对BCR-CDR3免疫组库的特征并不清楚。近年，第二代高通量测序技术（NGS）为描绘免疫组库的全貌提供了强有力的技术支撑。本项目将采用arm-PCR技术特异性扩增PBC患者外周血BCR-CDR3区核苷酸序列，通过NGS技术进行测序，结合生物信息学分析PBC患者BCR-CDR3多肽构成及频率，了解其免疫系统多样性的特点，寻找疾病特异性CDR3多肽序列或特征谱，为探索PBC发病机制、疾病个体化诊断及疗效评估提供新的思路。

原发性胆汁性胆管炎（PBC），又名原发性胆汁性肝硬化，是一种器官特异性自身免疫性肝脏疾病，免疫学特点是血清中出现抗线粒体抗体（AMAs）及自身免疫介导的肝内中小胆管损伤，发病机制尚不明确。B细胞与PBC患者非化脓性胆管炎及胆管损伤的免疫机制有关，其抗原受体B细胞受体（BCR）重链CDR3区，即BCR免疫球蛋白重链互补决定区3（IGH-CDR3）变异程度最高，构成容量巨大的免疫组库，赋予个体识别各种抗原、产生特异性抗体的巨大潜能。早期由于受研究技术所限，人们对PBC患者BCR多样性的认识并不全面，对BCR-CDR3免疫组库的特征并不清楚。近年，第二代高通量测序技术（"Next-generation" sequencing technology，NGS）为描绘免疫组库的全貌提供了强有力的技术支撑。本项目采用arm-PCR技术特异性扩增35例PBC患者IGH-CDR3区核苷酸序列，通过NGS技术进行测序，结合生物信息学分析了PBC患者个体水平和群体水平IGH-CDR3多肽构成及频率，了解了其免疫系统多样性的特点。研究结果如下：1. 在个体水平，个别PBC患者IGH-CDR3免疫组库多样性程度较低，存在个别克隆异常增殖情况；2. 在群体水平，35例患者平均每个人测得序列数是414317.9714，总CDR3序列为409479.9714，独特CDR3序列为54694.6571；3. 通过与20例健康对照个体比较，PBC患者IGH-CDR3多样性参数虽然低于健康对照，但差异并无统计学意义；4.目前尚未发现PBC特异性IGH-CDR3多肽序列。本研究采用arm-PCR结合NGS以及iRmap软件分析的策略初步探索了PBC患者外周血B细胞IGH-CDR3免疫组库特征，为探索发病机制、以及疾病个体化诊断及疗效评估提供了新的思路，但距临床应用仍需群体大数据的积累。