建立九色流式检测人外周血中TCR Vβ淋巴细胞亚群活化及凋亡方案  

Using 9-color Flow Cytometry Immunophenotyping to Detect Activation and Apoptosis of Human TCR Vβ Lymphocyte Subpopulations in Peripheral Blood Samples

作　　者：黄巧容[1] 温博[2] 李雪[1] 胡莲[1] 万雪梅[2] 曾升平[3] 孟文彤[1] 

机构地区：[1]四川大学华西医院干细胞生物学研究室,成都610041 [2]成都中医药大学,成都610075 [3]成都中医药大学附属医院,成都610072

出　　处：《四川大学学报（医学版）》2016年第2期262-266,共5页Journal of Sichuan University(Medical Sciences)

基　　金：国家自然科学基金国际(地区)合作交流项目(No.81361120381)资助

摘　　要：目的建立九色流式方法检测人外周血中TCR Vβ淋巴细胞亚群活化及凋亡状态的方案。方法用细胞膜表面抗体CD3、CD4、CD8、CD95、CD69、磷脂结合蛋白Annexin-Ⅴ、TCR Vβ抗体试剂盒和细胞核染料DAPI建立九色流式染色方案。用1例健康成年人外周血标本分别进行抗体滴度实验、最适检测电压选择、单色染色和缺一色染色,确定条件后,对8例健康成年人外周血标本进行检测分析。结果根据细胞分群情况选择了合适的抗体滴度和检测电压,单色染色和缺一色试验确定了适宜补偿,建立了九色流式检测人外周血中TCR Vβ淋巴细胞亚群活化及凋亡状态的方案;采用此方案检测的8例健康成年人标本,TCR Vβ亚群分布与TCR Vβ抗体试剂盒说明书及文献报道分布一致,并能分析各TCR Vβ亚群细胞活化及凋亡状态。结论建立的九色流式方案可检测人外周血中TCR Vβ淋巴细胞亚群活化及凋亡状态,操作简便,结果可靠。Objective To establish an assay using 9-color flow cytometry immunophenotyping to detect activation and apoptosis of human TCR Vβlymphocyte subpopulations in peripheral blood samples.Methods We used 5 antibodies（CD3,CD4,CD8,CD95,CD69）,phospholipids binding proteins Annexin Ⅴ,TCR VβRepertoire Kit and nucleus dye DAPI to establish a 9-color flow cytometry assay.Peripheral blood samples were taken from eight healthy people for test of antibodies and determination of optimal PMT and staining method（single-stained vs stained with all but one antibody）. Results Appropriate detecting voltage,antibody concentration and compensation methods were determined.The distribution of TCR Vβsubgroup in our samples was consistent with the TCR Vβ Repertoire Kit instruction and other published literature.Conclusion We have established a effective easy using 9-color flow cytometry immunophenotyping to detect human TCR Vβlymphocyte subpopulations in peripheral blood samples.

关 键 词：九色流式 T细胞亚群 TCR VΒ 活化 凋亡 

分 类 号：R392[医药卫生—免疫学]