流式技术测定乙型肝炎患者外周血特异性CD8＋T细胞及其临床意义

Determination of circulating HBV specific CD8 ＋ T cells in hepatitis B patients by flow cytometry and its clinical significance

目的 探讨流式细胞技术检测乙型肝炎患者HBV特异性CD8＋T细胞的水平并分析其临床意义.方法 采用HLA-A2分子胞外段与HBV核心表位肽core18-27结合的HLA-肽五聚体（Pentamers）对25例急性乙肝患者、35例慢性乙肝患者和10例正常健康人的外周血进行染色,设计流式细胞技术检测其针对该肽段的特异性CD8＋T细胞数量,以占总计数CD8＋细胞数的百分比表示.结果 12例HLA-A2＋的急性乙肝患者急性期可检测到高水平的特异性CD8＋T细胞,中位数为2.93%（1.12%～4.63%）,16例HLA-A2＋的慢性乙肝患者特异性CD8＋T细胞水平较低,中位数为0.75%（＜0.01%～1.76%）,两组之间差异有统计学意义（P＜0.01）.10例HLA-A2＋正常对照组、13例HLA-A2-急性乙肝对照组和19例HLA-A2慢性乙肝对照组的特异性CD8＋T细胞均不超过0.02%.结论 HLA-肽五聚体流式细胞技术能在体外直接检测HBV特异性的CD8＋T细胞数量,其水平可能影响着HBV感染者体内病毒的清除,且与乙肝的不同临床转归有关.

Objective To explore the levels of circulating HBV specific CD8＋ T cells in hepatitis B patients and analysis its clinical significance. Methods HBV specific CD8 ＋ T cells in whole blood samples of twenty-five acute hepatitis B patients, thirty-five chronic hepatitis B patients and ten healthy control were stained with pentamers complex of HLA-A2 and HBV core 18-27 peptide and counted by flow cytometry.Results The medians of HBV core 18-27 specific CD8＋ T cells quantities among total CD8＋ cells were 2.93% （ 1.12% -0. 63% ） in 12 HLA-A2 ＋ acute hepatitis B patients and 0. 75% （ ＜ 0. 01% - 1.76% ） in 16 HLA-A2＋ chronic hepatitis B patients respectively. There was showed a marked significant between the two groups （P ＜ 0. 01 ）. The medians of HBV core 18-27 specific CD8＋ T cells of 10 HLA-A2＋ healthy control, 13 HLA-A2- acute hepatitis B patients and 19 HLA - A2- chronic hepatitis B patients were all lower than 0. 02% separately. Conclusion HLA-peptides pentamers staining flow cytometry is a direct ex vivo method to detect the levels of circulating HBV specific CD8 ＋ T cells which may be play a crucial role in complete clearance of HBV and relates to clinical consequence in hepatitis B patients.