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目的 建立原发性胆汁性肝硬化(PBC)患者外周血CD4+T细胞受体(TCR) Vβ链的CDR3免疫组库,分析其多样性的改变及TCR V β链CDR3序列的优势取用情况.方法 利用扩增子救援多重PCR (Arm-PCR)技术和高通量测序技术半定量扩增并测序得到CD4+T细胞TCR Vβ链的CDR3序列,并结合临床资料对测序结果进行生物信息学分析.两组间均数比较用非参数检验. 结果 成功建立了7例PBC患者外周血CD4+T细胞TCR Vβ链CDR3免疫组库;PBC患者组CDR3序列多样性为11.3±2.9 (D50值,x±s,n=7)低于健康志愿者组的18.1±0.6 (D50值,n=10)(P＜0.05),且晚期PBC患者CDR3序列多样性(4.5±2.9)较早期患者CDR3序列多样性(16.7±2.8)降低(P＜0.05);7例PBC患者外周血CD4+T细胞均共有的TCR Vβ链CDR3优势取用序列多达52种,通过与健康志愿组进行比较,初步筛选出4种优势取用氨基酸序列“ASSFTGGPVEQY“ “ASSLISSGNNEQF” “ATSRDTLAGGPGDTQY” “SASLEGNTEAF”,且4种PBC优势取用序列在晚期PBC患者中出现的频次较早期显著增加.结论 本研究通过7例PBC患者外周血CD4+T细胞TCR Vβ链CDR3免疫组库的建立及多样性和疾病优势取用序列的分析,提示特征性(具有特定TCR VβCDR3序列)的CD4+T细胞大量克隆扩增可能是该疾病发生及进展的重要因素,为临床免疫治疗提供了重要的理论基础和分子靶点.“,”Objective To determine the immune repertoires of peripheral CD4+ T cell receptor (TCR) Vβ CDR3 in primary biliary cirrhosis (PBC) and analyze TCR diversity and preferred usage at sequence-level resolution.Methods ARM-PCR and high-throughput sequencing were used to obtain millions of TCR Vβ CDR3 sequences from peripheral CD4+ T cells isolated from 7 patients with PBC and healthy volunteers.All sequencing data were analyzed,together with corresponding clinical information,by bioinformatic software.The MannWhitney U test was used for statistical analysis.Results The PBC patients showed a lower level of diversity among the peripheral CD4+ TCR Vβ CDR3 than the healthy volunteers,and patients with higher level progression of the disease showed a greater lack of diversity.In addition,4 specific preferred-usage amino acid sequences were discovered for the PBC patients:ASSFTGGPVEQY,ASSLISSGNNEQF,ATSRDTLAGGPGDTQY,and SASLEGNTEAF;these sequences were also found in higher frequencies in patients with later stages of PBC.Conclusions Decreased TCR Vβ CDR3 diversities and specific preferred usage of TCR CDR3 sequences in peripheral CD4+T lymphocytes in PBC suggest that clonal expansion of a large number of CD4+T cells may be an important factor for PBC progression.These data provide a better understanding about the general characteristics of CD4+T cells in PBC patients and related to pathogenesis of the disease,and may provide useful insights into potential targets for immunotherapy.