IFN-α治疗后发生HBsAg转阴的慢性乙型肝炎患者外周血免疫库谱特征分析

Analysis of immunological repertoire in the peripheral blood of chronic hepatitis B patients with clearance of HBsAg after IFN-α treatment

目的通过对慢性乙型肝炎患者经IFN-α治疗后出现HBs Ag转阴病例与未转阴病例外周血中T细胞受体β链(TCRβ,TRB)序列分析比较,探讨HBs Ag转阴病人外周血免疫库TCRβ的特征,为预测病人临床结局以及评价治疗效果提供新的指标。方法收集HBV感染病人的外周血样进行密度梯度离心获得淋巴细胞,冻存。待IFN-α治疗一段时间后,取HBs Ag转阴病例与未转阴病例相应的冻存细胞,提取RNA,逆转录为c DNA后通过多重PCR对TCRβ链的CDR3区序列进行扩增、纯化,高通量测序后将测序结果与IMGT数据库进行比对,以找到HBs Ag转阴病人外周血TRB-CDR3免疫组库的特征,分析人外周血TRB-CDR3多样性及其他免疫学特征与HBs Ag转阴之间的关系。结果 IFN-α治疗后HBs Ag转阴的患者TRB-CDR3的多样性高于未转阴患者,并表现出其特有的免疫学特征,包括高频取用的V、J家族、V-J家族配对、基因的插入、CDR3区长度分布等均与未转阴组有明显差异,而基因的缺失方面未发现差异。结论 IFN-α治疗后HBs Ag转阴的患者的免疫组库呈现明显的免疫学特征,可为临床疗效的预测提供新的切入点。

To find the characteristics of the immunological repertoire in the peripheral blood of chronichepatitis B patients with clearance of HBs Ag after IFN-α treatment, we collected peripheral blood of chronic hepatitisB patients to get the lymphocytes by density gradient centrifugation. After IFN-α treatment, the patients with orwithout clearance of HBs Ag were selected, and their corresponding lymphocytes were picked up for total RNAextraction. Then the total RNA was reverse transcribed to c DNA, and the CDR3 sequence of TCRβ was amplified,which was compared with IMGT database then analyzed. Data showed that the diversity of CDR3 of the peripheralblood T cell was lower in chronic hepatitis B patients with clearance of HBs Ag as compared with chronic hepatitis Bpatients without clearance of HBs Ag. Furthermore, there were also difference in other immunological features, suchas the usage of V/J gene, the pair of V and J gene, the insertion of gene and CDR3 length, except the deletion ofgene. So, we can draw a conclusion that chronic hepatitis B patients with clearance of HBs Ag after IFN-α treatmentdisplay distinct immunological repertoires, which couldbe used in clinical outcome prediction of chronichepatitis B patients.