乙型肝炎病毒性相关性肝细胞癌患者外周血与肝癌组织T淋巴细胞受体谱系分析

Analysis of T cell receptor repertoire diversity in peripheral blood lymphocytes and tumor-infiltrating T lymphocytes of patients with hepatitis B Virus related hepatocellular carcinoma

目的比较乙型肝炎病毒性相关性肝细胞癌（HBV-RHCC）患者外周血T淋巴细胞（PBLs）与肝癌组织中浸润性T淋巴细胞（TILs）受体（TCR）谱系及互补决定区3（CDR3）序列特点。方法选择新乡市中心医院2014年3～8月行肝癌切除术患者10例为研究对象。应用反转录-聚合酶链反应（RT-PCR）扩增TCRβ链可变区（BV）中CDR3基因的24个家族,采用扭曲率、缺失率、正态率、单峰率以及复杂评分分析TCR-BV各家族谱系的完整性与多样性,并将优势利用的谱系家族进行CDR3测序。结果 10例HBV-RHCC患者CDR3谱系多数呈现扭曲分布。PBLs与TILs复合评分比较差异有统计学意义（P=0.042）;且上述谱系特征与肝癌家族史、AFP水平有关,但与患者的病毒载量无关。此外,在PBLs与TILs中发现了＂GGTGVSPLH＂＂LGTGNDDPF＂共有序列。结论 HBV-RHCC患者PBLs与TILs中存在克隆性增生,并证实了局部细胞免疫功能差异的存在。多个共有序列的发现,为HBV-RHCC的靶向治疗提供理论基础与潜在的靶点。

Objective To compare the feature of T cell receptor（TCR） repertoire diversity and complementarity deter- mining region3 （CDR3） sequences in peripheral blood lymphocytes （PBLs） and tumor-Infiltrating T lymphocytes （TILs） of the patients with hepatitis B Virus related hepatocellular carcinoma（HBV-RHCC）. Methods Ten patients with hepateetomy in the Central Hospital of Xinxiang City from March to August 2014 were selected. TCR-BV families were amplified by reverse transcription polymerase chain reaction and the repertoire diversity of BV families. Twist rate, miss rate, normal distribution rate, single rate and complexity scores were used to evaluate the integrity and diversity of TCR profile between PBLs and TILs. The clonally of the T cells was further analyzed by CDR3 sequencing. Results A majority of the CDR3 profiles of PBLs and TILs in 10 HBV-RHCC patients showed a distorted distribution. There was significant difference in the complexity score of the profiles in TILs compared with PBLs compartment （ P = 0. 042 ）. This pattern was respective related to family history of hepato- cellular carcinoma and level of alpha-fetoprotein, with the exception of the patient＇s HBV - DNA viral load. In addition, the consensus sequences ＂GGTGVSPLH＂ and ＂LGTGNDDPF＂ were found distributed between PBLs and TILs compartments in different patients. Conclusions The study find an apparent clonal proliferation in PBLs and TILs compartments, and con- firmed a remarkable difference in cellular immunology in PBLs and TILs. The relatively conserved amino acid motifs provide fundamental basis and could be a potential target for targeted therapy in HBV-RHCC patients.