应用CD4^+TCR四聚体-流式分析和细胞爬片法检测分析外周血结核抗原特异性CD14^+单核细胞  

Detection of antigen-specific CD4^+ monocytes in blood of tuberculosis patients by using CD4^+ T-cell receptor tetramer-based flow cytometric analysis and cell climbing slice assay

作　　者：吴荣顺[1,2] 赖小敏[2] 谢丹[1,2] 方毅敏[3] 张扣兴[1] 

机构地区：[1]中山大学附属第三医院重症医学科,广州510630 [2]中山大学中山医学院 [3]广州市胸科医院

出　　处：《中国感染与化疗杂志》2014年第6期503-507,共5页Chinese Journal of Infection and Chemotherapy

基　　金：国家自然科学基金面上项目(81271779);十二五传染病防治重大科技专项分任务(2012ZX10004903-004-002)

摘　　要：目的探讨CD4+TCR四聚体-流式分析和细胞爬片法检测分析外周血结核抗原特异性CD14+单核细胞的应用价值。方法应用四聚体对肺结核患者(肺结核组)外周血进行染色,流式检测四聚体阳性CD14+细胞的百分率,以非结核呼吸道感染患者(非结核呼吸道感染组)外周血及脐带血标本(脐带血组)作为研究对照。将肺结核组和对照组外周血制作成细胞爬片,用四聚体-细胞爬片法原位染色,激光共聚焦倒置显微镜观察细胞爬片中四聚体与结核抗原双染阳性及四聚体与CD14双染阳性细胞的分布情况。结果应用CD4+Vα21-J39/Vβ29-D1-J2 TCR四聚体检测肺结核组、非结核呼吸道感染组和脐带血组的四聚体阳性CD14+细胞百分率中位数分别为1.32%、0.50%和0.26%,而CD4+Vα21-J39/Vβ29-D2-J2 TCR四聚体则分别为1.05%、0.49%和0.19%。肺结核组外周血CD14+单核细胞百分率显著高于各对照组。细胞爬片法可观察到肺结核组四聚体与CD14双染阳性及四聚体与结核抗原双染阳性的细胞,而对照组标本均为阴性。结论 CD4+TCR四聚体技术,结合细胞流式和细胞爬片原位染色方法,能在体外敏感地检测出结核抗原特异性CD14+单核细胞,可以更准确地评价其在结核感染者体内的变化特征及临床意义。Objective To evaluate the utility of CD4^＋ TCR tetramers‐based flow cytometric analysis and cell climbing slice assay in detecting antigen‐specific CD14＋ monocytes in the blood of tuberculosis （TB） patients .Methods CD4^＋ TCR tetramers were used to detect tetramer‐positive CD14＋ monocytes in the peripheral blood （PBL ） samples of inpatients with advanced pulmonary TB （PTB） by flow cytometric analysis .The PBL samples obtained from non‐TB patients and umbilical cords were used as controls .These tetramers were also used to examine tetramer‐bound CD14＋ monocytes and Mycobacterium tuberculosis （MTB） antigen‐specific and tetramer‐bound cells by cell climbing slice in situ staining .Results The median percentage of＆amp;nbsp;tetramer‐bound CD14＋ monocytes in PBL samples from PTB patients ,non‐TB patients and umbilical cords were 1 .32% , 0 .50% and 0 .26% respectively by using CD4^＋ Vα21‐J39/Vβ29‐D1‐J2 tetramer , while the medians were 1 .05% , 0 .49% and 0 .19% respectively by using CD4^＋ Vα21‐J39/Vβ29‐D2‐J2 tetramer . The percentage of tetramer‐bound CD14＋ monocytes in PTB patients group was significantly higher than the other two control groups .In cell climbing slice in situ staining ,tetramer‐bound CD14^＋ monocytes ,and MTB antigen‐specific and tetramer‐bound cells were positive in PTB tissue compared with negative in control tissues . Conclusions CD4＋ TCR tetramers‐based flow cytometric analysis and cell climbing slice assay could be used to sensitively detect MTB antigen‐specific CD14^＋ monocytes in the blood of TB patients ,and more accurately evaluate the changing profile and clinical significance of these cells in TB patients .

关 键 词：肺结核 CD4^+ TCR四聚体 CD14^+单核细胞 

分 类 号：R392[医药卫生—免疫学]