CFSE标记人T细胞亚群增殖反应及其在病毒感染性疾病中的应用  

Application of the proliferative response of human T lymphocyte subsets labeled by CFSE in virus infectious diseases

作　　者：彭巧丽[1] 李海英[1] 曹振环[1] 田亚坤[1] 陈新月[1] 吴昊[1] 计云霞[1] 

机构地区：[1]首都医科大学附属北京佑安医院,北京100069

出　　处：《中国病原生物学杂志》2008年第12期881-885,共5页Journal of Pathogen Biology

基　　金：北京市自然科学基金资助项目(No.5062019);北京市科委艾滋病重大项目基金赞助(No.D0906003040591);国家高技术研究发展计划(863计划)(No.2006AA02Z411)

摘　　要：目的探讨应用流式细胞术和活体染料羧基荧光素二乙酸盐琥珀酰亚胺酯(CFSE)标记人T细胞亚群增殖反应的方法学及其在病毒感染性疾病中的应用价值。方法用不同浓度的CFSE标记新鲜分离的人外周血单个核细胞(PBMC),分为未刺激组及刺激组。未刺激组加入CD28,刺激组加入CD28及不同浓度PHA,将细胞培养5～7d后进行染色,采用流式细胞仪检测,观察CFSE、PHA标记的最佳浓度及最佳培养时间,采用CellQuest软件及ModFit软件分析T淋巴细胞各亚群的增殖情况,并对两种分析方法进行比较,在上述最佳实验条件下,采用相同的实验方法分析CMV-pp65肽刺激CMV-IgG(+)HLA-A2(+)者外周血单个核细胞(PBMC)后T细胞亚群增殖情况;结果CFSE标记人外周血单个核细胞(PBMC)的最佳浓度是0.5μmol/L,PHA的最佳刺激浓度为2μg/ml,在此浓度下,最佳培养时间为6d,PHA刺激培养6d后T细胞各亚群出现典型的增殖不同步现象,经CMV-pp65肽刺激后CD8+T细胞出现明显的增殖反应,采用CellQuest及ModFit软件联合分析,可实现淋巴细胞增殖分析的可视化及数量化。结论CFSE染色结合荧光抗体标记和流式细胞术是分析淋巴细胞增殖的有力工具,可以有效检测出病毒抗原刺激后T淋巴细胞各亚群的增殖反应及增殖动力学,在探讨病毒感染性疾病的发病机制中具有重要价值。Objective To study the methodology of combining carboxyfluorescein diacetatesuccinimidyl ester （CFSE） with flow cytometry to label the proliferitive response of human T lymphocyte subsets and its application in virus infectious diseases. Methods To select the optimal concentration and culture time, the fresh PBMC isolated from healthy subjects were labeled with CFSE on different concentrations, divided into unstimulated group and stimulated group, one group added with CD28, the other group added with CD28 and PHA, and incubated at different time from 5 to 7 days, then stained and determined by flow cytometry.The proliferative dynamics of human T cell subsets were analyzed with CellQuest and ModFit software.At the above optimal experiment conditions, the proliferative situation of T lymphocyte subsets from CMV-IgG positive and HLA-A2 positive subjects after simulated with CMV-pp65 peptide were analyzed. Results The optimal labeling concentration was 0.5 μmol/L for CFSE and 2 μg/ml for PHA.Under these conditions, six day was selected as the optimal culture time.The proliferation of T lymphocyte subsets were atypically asynchronous after stimulated with PHA, and the CD8＋T cells showed obvious proliferation after simulated with CMV-pp65 peptide.The combination of CellQuest with ModFit software can realize the quantification and visualization in analyzing the proliferation of lymphocyte. Conclusion CFSE staining combined with fluorescent antibody labeling and cytometry are powerful tools for analysis of lymphocytic Proliferation.It can effectively detect the proliferation kinetics of T lymphocyte subsets after stimulated with virus antigen.It will be of great value in exploring the pathogenesis of virus infectious disease.

关 键 词：T淋巴细胞 CFSE 增殖反应 

分 类 号：R511[医药卫生—内科学]