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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 investigate the response of specific cytotoxic T lymphocytes (CTL) in peripheral blood of patients with severe hepatitis B by using the technique of Tetramer flow cytometry (MHC) Its clinical significance. Methods Tetramer flow cytometry was used to detect the number of three types of CD8 + CTL cells restricted by human leukocyte antigen (ILE) in peripheral blood of patients with severe type B hepatitis B. By using enzyme-linked immunosorbent assay (ELISA) Hepatitis B virus (HBV) peptide-induced culture of specific CTL expression of membrane cytokines such as IFNγ, TNFα, IL 4 and IL-10 and other levels; using Promega CytoTox96 non-radioactive cytotoxicity test technique, determined by specific HBV peptide induction Specific CTLs are cultured to kill target cells. Results The number of CTLs specific to HBVcore18-27 in the peripheral blood of patients with acute severe hepatitis B was higher than that of patients with chronic severe hepatitis B (P <0.05) and lower than that of patients with acute hepatitis B (P <0. 05). The levels of IFN-γ and tumor necrosis factor-α in patients with acute severe hepatitis B were higher than those in patients with chronic severe hepatitis B (all P <0.05). The HBV core 18-27 specific CTLs in patients with acute severe hepatitis B The target cell capacity was higher than that of chronic severe hepatitis B group (P <0.05). Conclusion The specific CTL response in patients with acute severe hepatitis B is enhanced, while the specific CTL response in patients with chronic severe hepatitis B is deficient. Peripheral blood specific CTL persisted in patients with acute severe hepatitis B, which may be related to the promotion of virus clearance.