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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。
OBJECTIVE: To compare the sequence analysis of TCRβ receptor (TRB) in peripheral blood of HBsAg-negative patients after chronic hepatitis B treated with IFN-α, The characteristics of TCRβ in blood immune library provide new indexes for predicting the patient’s clinical outcome and evaluating the therapeutic effect. Methods Peripheral blood samples from patients with HBV infection were collected for density gradient centrifugation to obtain lymphocytes. After treatment with IFN-α for a period of time, the corresponding cryopreserved cells of HBsAg-negative and non-negative cases were taken, RNA was extracted, reverse transcribed into c DNA, and the CDR3 sequence of TCRβ chain was amplified by multiplex PCR and purified , The sequencing results were compared with IMGT database after high-throughput sequencing to find out the characteristics of TRB-CDR3 in the peripheral blood of HBsAg-negative patients. The diversity of TRB-CDR3 in human peripheral blood and other immunological features were compared with HBsAg Ag negative relationship between the negative. Results The diversity of TRB-CDR3 in HBsAg-negative patients after IFN-α treatment was higher than that in non-negative patients and showed its unique immunological characteristics including VJ family, VJ family pairing, The gene insertion, CDR3 region length distribution and so on were significantly different from the non-negative group, but no difference in gene deletion. Conclusion The immunological characteristics of patients with negative HBsAg after IFN-α treatment show obvious immunological characteristics, which may provide a new entry point for the prediction of clinical efficacy.