流式细胞仪三色法检测外周血Th细胞亚群培养方案的研究  

Study of Culture Project for Three-Color Detection of Th-Subgroup in Peripheral Blood by Flow Cytometry

作　　者：叶卫东[1] 张循善[2] 翟志敏[3] 

机构地区：[1]武警安徽总队医院检验科,安徽合肥230041 [2]安徽医科大学第一附属医院输血科,安徽合肥230032 [3]安徽省立医院中心实验室,安徽合肥230001

出　　处：《现代检验医学杂志》2006年第4期27-29,共3页Journal of Modern Laboratory Medicine

基　　金：安徽省教育厅科研基金资助项目(编号:2001kj147)

摘　　要：目的改进流式细胞仪三色法检测外周血T h细胞亚群的刺激培养方案。方法外周全血经RPM I1640培养液和终浓度为25μg/L的PM A、1 m g/L的Ionom yc in、20μg/L的M onens in培养2 h后,用流式细胞仪分别检测CD 4、CD 69阳性细胞及CD 3阳性细胞表达IFN-γ和IL-4的比例。同时检测培养前CD 4阳性细胞及培养4 h后CD 3阳性细胞表达IFN-γ和IL-4的比例。结果淋巴细胞门中CD 4+细胞与CD 4-细胞能明显区分,培养前与培养2 h后CD 4+细胞比例差异无显著性(P>0.05);T淋巴细胞中90%以上表达CD 69;刺激培养2 h和4 h后CD 3+细胞表达IFN-γ和IL-4的比例差异无显著性(P>0.05)。结论该实验确定的培养方案能满足流式细胞术CD 4、IFNγ-、IL-4三色法检测T h细胞亚群的培养要求。Objective To improve a culture project for three-color detection of Th subgroup in peripheral blood by flow cytometry. Methods Peripheral whole blood was co-cultured with RPMI1640,25μg/L of PMA, lmg/L of Ionomycin and 20μg/L of Monensin for 2 hours,then the percentages of CD4^＋ cells,CD69^＋cells,IFN-γ^＋of CD3 cells and IL-4^＋ of CD3 cells were detected by flow cytometry respectively. The percentages of CD4^＋cells before cultured,IFN-γ^＋f CD3 cells and IL-4^＋of CD3 cells after cultured for 4 hours were detected by flow cytometry. Results CD4^＋ cells and CD4^-cells can be discriminated by CD4 molecular of cell surface in lymphocyte gate. Compared with before cultured,the percentage of CD4^＋ cells after cultured for 2 hours was no difference （P〉0. 05）. CD69 were expressed by more than 90 percent of T cells.The percentages of IFN-γ^＋of CD3 cells and IL-4^＋ of CD3 cells cultured for 2 hours or 4 hours were no difference （P〉0. 05） . Conclusion The culture project can meet the requisition of CD4,IFN-γ and IL-4 three-color method for detecting Th subgroup in peripheral blood by flow cytometry.

关 键 词：培养方案 流式细胞仪 三色法 T辅助细胞亚群 

分 类 号：R446.61[医药卫生—诊断学]