乙型肝炎患者治疗前后外周血CD4^+CD25^+Tregs TCR CDR3谱系漂移特征分析  

Spectrum drift characteristics of CD4^+CD25^+Tregs TCR CDR3 in hepatitis B patients before and after treatment

作　　者：潘宗琴[1] 吕红[1] 庄勤建[1] 姚新生[2] 邱隆敏[1] PAN Zong-Qin LU Hong ZHUANG Qin-Jian YAO Xin-Sheng QIU Long-Min(Department of Infection, Zunyi Medical College Affiliated Hospital, Zunyi 563003, Chin)

机构地区：[1]遵义医学院附属医院感染科,遵义563003 [2]遵义医学院免疫学教研室,遵义563003

出　　处：《中国免疫学杂志》2017年第6期889-894,899,共7页Chinese Journal of Immunology

基　　金：贵州省社会发展公关基金资助项目(No.黔科合SY[2010]3049号)

摘　　要：目的:探讨AHB患者急性期及恢复期、CHB患者恩替卡韦治疗前后外周血PBMC中CD4^+CD25^+Tregs TCR CDR3谱系漂移特征的变化。方法:采集4例正常人、3例AHB患者(急性期和恢复期)及4例CHB患者恩替卡韦治疗前后外周抗凝静脉血,分离外周血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)设计一条共用的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软件分析研究对象TRBV家族CDR3谱系特征;采用配对t检验(paired-sample t test)检测AHB患者急性期及恢复期、CHB患者恩替卡韦治疗前后TRBV家族谱系漂移率差异。结果:3例AHB患者急性期呈现普遍谱系漂移的TRBV4、10、14、16、19家族在恢复期多转变为正态的多峰谱型,AHB患者恢复期外周血CD4^+CD25^+Tregs TRBV家族CDR3谱型漂移率显著低于急性期(t=9.456,P=0.011);TRBV8、11、13.2、15、16、18、20在3例CHB患者抗HBV治疗前出现克隆增生,TRBV1、5.2、6、12、14、24在3例CHB患者抗HBV治疗后出现克隆增生。CHB患者抗病毒治疗后TRBV家族CDR3谱型漂移率高于治疗前(t=-0.666,P=0.553)。结论:AHB患者急性期呈现谱系漂移的TRBV4、10、14、16、19家族在恢复期多转变为正态的多峰谱型,推测这种转变可能与HBsAg、HBeAg转阴相关。CHB患者在治疗前TRBV8、11、13.2、15、16、18、20家族克隆增生的Tregs可能通过下调机体的细胞免疫应答,阻碍病毒清除;随着药物导致病毒载量的显著下降,TRBV1、5.2、6、12、14、24家族克隆增生的Tregs可能更有助于诱导机体出现免疫耐受,导致HBV不能彻底清除。Objective：To determine the spectrum drift characteristics of CD4＋ CD25 ＋Tregs TCR β chain CDR3 in patients with different phases of acute hepatitis B （AHB） and chronic hepatitis B （CHB） patients before and after the entecavir treatment. Methods： Anticoagulation venous blood was collected from 4 normal control subjects,3 AHB patients with acute phase and convalescent phase, and 4 CHB patients before and after the entecavir treatment;and peripheral blood mononuclear cells were isolated; CD4＋ CD25＋ Tregs were separated by using the magnetic beads, and total RNAs were extracted from CD4＋ CD25＋ Tregs and used for reverse transcription. The TRBV CDR3 was amplified by polymerase chain reaction （PCR） with forward primers specific for 24 TRBV families and one fluorescence-labeled common reverse primer specific for the BC region. The PCR products were sent out for Genescan, and results were analyzed for the TRBV family CDR3 spectrum characteristics by using the Peak Scanner Software vl. 0. Data were analyzed with the comparative t-test to perform the statistical analysis. Results： The CDR3 spectral types of the TRBV family showed drift charac- teristics in 3 cases of AHB patients with acute and convalescent phases;single/oligo peak spectral type family was observed in most of patients with acute phase;multiple peak spectral type was seen in patients with convalescent phase;and the common spectrum shift of TRBV4,10,14,16,19 families seen in patients with acute phase was changed to multiple peak spectral type. The clonal expansion of TRBV family in the CD4＋CD25＋Tregs in PBMC from AHB patients with convalescent phase was significantly lower than AHB patients with acute phase （ t = 9. 456, P = 0.011 ）. The clonal expansion of Tregs TRBV13.2,15,16,18,20 family seen in CHB patients before treatment may interfere the virus removal through down-regulating the body＇s immune response; and with the decline of viral load in serum after the antiviral treatment,the clonal expansion of Tregs TRBV1

关 键 词：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 调节性T细胞 恩替卡韦 

分 类 号：R392-33[医药卫生—免疫学]