不同HBV感染状态患者外周血CD4^+CD25^+Tregs TCR CDR3谱系漂移特征分析  

The spectrum drift characteristics of CD4^+CD25^+Tregs TCR CDR3 in different HBV-infected patients

作　　者：吕红[1,2] 潘宗琴[1] 陈宇[1] 庄勤建[1] 姚新生[3] 邱隆敏[1] 

机构地区：[1]遵义医学院附属医院感染科,563003 [2]济宁市第一人民医院重症医学科,272000 [3]遵义医学院附属医院免疫学教研室,563003

出　　处：《免疫学杂志》2017年第8期683-690,共8页Immunological Journal

摘　　要：目的探讨慢性HBV携带者、慢性乙型肝炎(chronic hepatitis B,CHB)及急性乙型肝炎(acute hepatitis B,AHB)这3种不同HBV感染状态下患者外周血中CD4^+ CD25^+调节性T细胞(T regulatory cells,Tregs)T细胞受体(T cell receptor,TCR)β链各家族互补决定区3(complementarity-determining region 3,CDR3)谱系漂移特征。方法采集3例正常人、5例慢性HBV携带者、12例CHB及5例AHB患者外周抗凝静脉血,分离外周血单个核细胞(peripheral blood mononuclear cell,PBMC);磁珠分选法分选CD4^+ CD25^+Tregs,提取总RNA,逆转录合成c DNA;根据TCRβ链24个可变区基因(TRBV)家族设计相应的上游引物,并在β链恒定区(BC)设计1条共用的FAM荧光标记下游引物及对照引物。PCR扩增出24个包含完整CDR3区的TRBV家族的PCR产物,电泳鉴定目的片段;PCR产物送上海基康用毛细管电泳法进行基因扫描;用Peak Scanner Software v1.0软件分析TRBV家族CDR3谱系特征。结果不同HBV感染状态下24个TRBV家族CDR3谱系图均呈现一个或者多个形态不一、数量不等的单峰、寡峰、偏峰谱型,其中一些家族表达极低或缺失,在1.5%琼脂糖凝胶电泳图上多数家族于预测范围大小处呈现1条模糊条带,部分家族出现清晰条带或无条带;对不同HBV感染者间各TRBV家族CDR3谱系偏移率进行比较发现:慢性HBV携带者中BV7谱系偏移率明显高于CHB及AHB患者(P均<0.05),CHB患者中BV15谱系偏移率明显高于慢性HBV携带者及AHB患者(P均<0.05),AHB患者中BV23谱系偏移率明显高于CHB及慢性HBV携带者(P均<0.05)。CHB患者外周血CD4^+ CD25^+Tregs TRBV家族CDR3克隆增生总体异常率与HBV DNA载量呈正相关关系(r=0.576 5,P=0.049 8)。结论部分优势利用的TRBV家族可能在感染HBV后免疫耐受以及清除病毒中发挥重要作用。This study was performed to investigate the spectrum drift characteristics of CD4^＋ CD25^＋Tregs TCR β chain CDR3 in different HBV-infected patients, involving chronic HBV carriers, CHB and AHB patients.Anticoagulation venous blood samples of 10 ml were collected from 3 normal controls, 5 chronic HBV carriers, 12 patients with CHB and 5 patients with AHB, and magnetic beads were used to separate CD4^＋ CD25^＋Tregs. Total RNAwas extracted from CD4^＋ CD25^＋Tregs, then the total RNA was reversely transcripted to c DNA. The TRBV CDR3 wasamplified by polymerase chain reaction（PCR） using specific primers for 24 TRBV families and one BC region primer（labeled with fluorescence）. The PCR products were sent out for Genescan, then analysis of the TRBV family CDR3 spectrum characteristics was carried out using the Peak Scanner Software v1.0. The PCR products that showed a single peak（clonal expansion） were amplifiedagain at the same PCR condition, but the BC region primer was not labeled by fluorescence. Data showed that in 3 normal controls, the spectrum of CD4^＋ CD25^＋Tregs TCR β chain CDR3 showed multiple peak spectral type（Gaussian distribution）, while the PCR products of 24 TRBV family showed a blur band in the prediction of products size on 1.5% agarose gel by Gold View staining. In different HBV-infected patients, the spectrum showed one or more single/oligo/partial peak spectral type, some patients even showed no TRBV family, while the PCR products of most of 24 TRBV family showed a blur band in the prediction of products size and parts of TRBV family showed either no or clear band on 1.5% agarose gel by Gold View staining.Furthermore, the skewing TRBV family in different HBV infection status was different. Comparison of the CDR3 spectrum drift rate among different patients showed that the spectrum drift rate of BV7 in chronic HBV carriers was significantly higher than that of CHB and AHB patients（P〈0.05）, the spectrum drift rate of BV15 in CHB patients was significantly higher t

关 键 词：慢性HBV携带者 慢性乙型肝炎 急性乙型肝炎 T淋巴细胞受体 互补决定区3 

分 类 号：R512.62[医药卫生—内科学]