基于免疫组库测序的肝硬化典型证候患者外周血T细胞受体β链CDR3多样性分析  

Diversity of the T Cell Receptor β Chain Complementarity-Determining Region 3 in Peripheral Bloodof Typical Syndromes Cirrhosis of the Liver: An Analysis Based on Immune Repertoire Sequencing

作　　者：王佳 胡宇婷 龚跃峰 朱健[3] 苏式兵 陆奕宇 WANG Jia;HU Yuting;GONG Yuefeng;ZHU Jian;SU Shibing;LU Yiyu(Shanghai University of Traditional Chinese Medicine Institute of Interdisciplinary Integrative Medicine Research,Shanghai 201203,China;Qingdong Hospital of TCM,Jiangsu 226200,China;Qidong Liver Cancer Institute,Jiangsu 226200,China)

机构地区：[1]上海中医药大学交叉科学研究院,上海201203 [2]江苏省启东市中医院,启东226200 [3]江苏省启东市肝癌防治研究所,启东226200

出　　处：《世界科学技术-中医药现代化》2024年第7期1916-1924,共9页Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology

基　　金：上海市“科技创新行动计划”自然科学基金项目(20ZR1453700):基于免疫组库测序和系统生物学动态网络分析的乙肝后肝硬化-肝癌证候演变机制研究,负责人:陆奕宇;国家自然科学基金委员会面上项目(82274183):基于免疫微环境中EZH2调控自然杀伤细胞探讨逍遥散及其成分组方改善肝纤维化的机制研究,负责人:陆奕宇。

摘　　要：目的 本研究通过免疫组库测序,分析肝硬化不同典型证候患者外周血TCR β链CDR3的多样性,探讨肝硬化证候的物质基础及其规律。方法 20例肝硬化患者为病例组,包括肝胆湿热(LGHD)、肝郁脾虚(LDSD)、肝肾阴虚(LKYD)3种证型,10例健康患者为正常对照组。从外周血样品中提取DNA,对TCRβ链CDR3进行多重PCR扩增,然后对产物进行高通量测序,分析其TCR β链CDR3的多样性。结果 肝硬化证候LDSD的CDR3独有nt序列数与CDR3独有aa序列数均少于LKYD(P<0.05);LGHD和LKYD的Clonality、Pielous、Shannon.Index和DE50具有显著性差异(P<0.05);3种证型的V和J区基因中多个片段使用频率及V-J基因重组有显著性差异:在LGHD和LDSD中,TRBV21-1、TRBV12-4、TRBV11-1亚型及7对V-J重组有统计学差异(P<0.05);在LGHD和LKYD中,TRBV10-2、TRBV7-6、TRBV5-8亚型及30对V-J重组有统计学差异(P<0.05);在LDSD和LKYD中,TRBJ1-5亚型及18对V-J重组有统计学差异(P<0.05)。结论本研究通过挖掘肝硬化不同证候的免疫学特征,发现肝硬化证候TCR CDR3的多样性具有显著差异且符合由实向虚的证型变化规律,为寻找“病证结合”“辨证论治”的客观依据提供新的支撑,以期发现肝硬化不同证候人群适应性免疫基因重排的表达差异和特异性标记。Objective In this study,the diversity of the TCRβchain CDR3 in peripheral blood of patients with different typical Traditional Chinese Medicine(TCM)syndromes of liver cirrhosis was analyzed by immune repertoire sequencing,the material basis and regularity of the syndromes of liver cirrhosis was discussed.Methods 20 patients with cirrhosis,including liver and gallbladder damp heat(LGHD),liver depression and spleen deficiency(LDSD),and liver and kidney yin deficiency(LKYD)were enrolled into case group,and 10 healthy patients were used as the healthy control group.DNA was extracted from peripheral blood samples,and multiplex PCR amplification of TCRβchain CDR3 was performed,followed by high-throughput sequencing of the products to analyze the diversity of the TCRβchain CDR3.Results The nt sequence numbers unique to CDR3 and aa sequence numbers unique to CDR3 of LDSD between liver cirrhosis syndromes were less than LKYD(P<0.05).Clonality,Pielous,Shannon.Index and DE50 of LGHD and LKYD had significant differences(P<0.05)between two groups,as well as the frequency of multiple fragments in V and J regions and V-J gene recombination of LGHD vs.LDSD,LGHD vs.LKYD,and LKYD vs.LDSD,respectively(P<0.05).LGHD vs.LDSD,TRBV21-1,TRBV12-4,TRBV11-1 subtype and 7 pairs of V-J recombination have statistical differences(P<0.05).LGHD vs.LKYD,TRBV10-2,TRBV7-6,TRBV5-8 subtypes and 30 pairs of V-J recombination were statistically different(P<0.05).LDSD vs.LKYD,there were statistical differences between TRBJ1-5 subtype and 18 pairs of V-J recombination(P<0.05).Conclusions The present study was conducted to find that the diversity of TCR CDR3 in liver cirrhosis syndrome is significantly different and conforms to the regularity of syndrome from excess to deficiency by explore the immunological characteristics of different TCM syndromes of liver cirrhosis,and to provide new support for the objective basis of"combination of disease and TCM syndrome"and"diagnosis and treatment".We explored the different expression patterns and specificity of

关 键 词：肝硬化 中医证候 免疫组库测序 T细胞受体 互补决定区3 

分 类 号：R285.6[医药卫生—中药学]