全血染色法和分离外周血单个核细胞染色的五聚体技术检测抗原特异性CTL的比较

Compare PBMC Staining and Fresh Whole Blood Staining Methods for Detecting Antigen-Specific Cytotoxic T Lymphocytes with MHC Pentamers

目的分别采用分离外周血单个核细胞(PBMC)法和全血直接染色法的组织相容性抗原-肽五聚体(MHCpentamers)流式细胞技术定量分析慢性乙型肝炎(下称慢性乙肝)患者体内乙型肝炎病毒(HBV)特异性的细胞毒性T淋巴细胞(CTL),并对两种方法进行比较。方法从慢性乙肝患者外周血中分离PBMC,用HLA-A2HBcAg抗原表位肽-MHCPentamers及CD8单克隆抗体染色后,用流式细胞仪检测HBV特异性的CTL细胞,部分患者同时采用全血直接染色方法进行HBV特异性的CTL细胞的检测。结果32例HLA-A2阳性的慢性乙肝患者外周血均可检测到HBV特异性的CTL细胞,五聚体阳性细胞占CD8阳性细胞的比例平均为(1.34±1.06)%,两种染色方法检测的特异性的CTL细胞无统计学差异。结论全血直接染色结合五聚体流式细胞技术检测抗原特异性CTL是一种简便、敏感和可行的抗原表位特异性CTL检测方法。

Objective To compare peripheral blood mononuclear ceils （PBMC） staining and fresh whole blood staining methods for detecting antigen-specific cytotoxic T lymphocytes （CTL） with MHC pentamers in chronic hepatitis B patients. Methods The frequency of HBcAg-specific CTL cells in PBMC of 32 patients with persistent HBV infection were quantified by flow cytometry using HLA-A2＊ HBV peptide pentamers. HBcAg-specific CTL cells of some samples were also detected through fresh whole blood staining. Results The HBcAg-specific CTL cells were detected in all individuals with chronic HBV infection. The average frequency of HBcAg-specific CTL cells was （1.34：1：1.06）%. There was no significant difference between PBMC staining and fresh whole blood staining methods. Conclusion The fresh whole blood staining is a convenient and sensitive method for detecting antigen specific CTL cells in HBV infection.