重型肝炎患者乙型肝炎病毒特异性细胞毒性T淋巴细胞应答功能研究

Detection of HBV specific cytotoxic lymphocytes responses in severe hepatitis B patients using MHC-peptide tetramer flow cytometry

目的用主要组织相容性复合物(MHC)-抗原肽四聚体(Tetramer)流式细胞技术,分析乙型重型肝炎患者外周血中特异性细胞毒性T淋巴细胞(CTL)应答状况,并探讨其临床意义。方法采用Tetramer流式细胞技术检测乙型重型肝炎患者外周血中受人类白细胞抗原I类分子限制的三类特异性CD8+CTL细胞数量;采用酶联免疫吸附斑点试验技术,测定经特异性乙型肝炎病毒(HBV)肽段诱导培养的特异性CTL表达膜内细胞因子IFNγ、TNFα、IL 4和IL-10等的水平;采用Promega CytoTox96非放射性细胞毒试验技术,测定经特异性HBV肽段诱导培养的特异性CTL杀伤靶细胞能力。结果急性乙型重型肝炎组外周血中针对HBVcore18-27表位的特异性CTL数量高于慢性乙型重型肝炎组(P<0．05),而低于急性乙型肝炎组(P<0．05);急性乙型重型肝炎组表达干扰素γ和肿瘤坏死因子α较慢性乙型重型肝炎组高(P值均<0．05);急性乙型重型肝炎组HBV core18-27特异性CTL裂解靶细胞能力高于慢性乙型重型肝炎组(P<0．05)。结论急性乙型重型肝炎患者特异性CTL应答作用增强,而慢性乙型重型肝炎患者特异性CTL应答缺乏。急性乙型重型肝炎患者外周血特异性CTL持续存在,可能与促进病毒清除等相关。

Objective To explore the role played by HBV antigen specific cytotoxic lymphocytes （CTLs） in severe hepatitis B patients. Methods Three kinds of HBV specific CD8^＋ T cells responding to HBV core 18-27, polymerase 575-583 and envelope 335-343 specific CD8^＋ T cells were searched with major histocompatibility complex （MHC）-peptide tetramer flow cytometry in the patients we studied. The cytokines produced by these specific CTLs such as IFN γ , TNF α, IL-4, IL-10 were detected using ELISPOT assays. Their ability to lyse target ceils was analyzed using a CytoTox96 Non-Radioactive Cytotoxicity Assay kit （Promega）. Results The number of HBV core 18-27 specific CD8^＋ T ceils was significantly higher in the peripheral blood of acute severe hepatitis B group patients than that in chronic severe hepatitis group （P 〈 0.05）, but lower than that in the acute hepatitis B patients. The IFN y and TNF α levels of acute severe hepatitis B patients were both significantly higher than those in the chronic severe hepatitis group （P 〈 0.05）. The ability to lyse target cells of HBV core 18-27 CTLs was also significantly higher in the acute severe hepatitis B group than that in the chronic severe hepatitis B group （P 〈 0.05）. Conclusion The response to antigen stimulation was much higher in acute severe hepatitis B than in chronic severe hepatitis B patients. The specific CTLs persisted among the peripheral blood mononuclear cells in acute severe hepatitis B, which may be related to the viral clearance.