流式细胞术检测淋巴细胞增殖方法的建立  

Establishment of flow cytometry assay of determination of lymphocytic proliferation

作　　者：李学义[1] 朱平[1] 樊春梅[1] 王彦宏[1] 史战国[1] 

机构地区：[1]第四军医大学西京医院临床免疫学,西安710032

出　　处：《中国免疫学杂志》2003年第1期43-46,共4页Chinese Journal of Immunology

摘　　要：目的 :建立一种用流式细胞仪同时测定T淋巴细胞增殖与细胞因子分泌的方法。方法 :用非特异性刺激剂PMA、PHA刺激 1 0例正常人外周血单个核细胞 ,体外培养 2、4、6、8、1 0h ,流式细胞术在单细胞水平检测T淋巴细胞的功能性激活、表面活化标志性抗原 (CD69)的表达、DNA合成 (BrdU掺入法 )及胞内细胞因子 (IL 4、IFN γ)分泌。选择合适的刺激剂、调整细胞浓度、优化实验方法 ,确定最佳实验条件。结果 :BrdU掺入法测定T淋巴细胞增殖 ,最佳的反应细胞浓度为 1× 1 0 6ml-1 ,比较不同刺激剂和体外培养时间 :PMA(2 0ng ml)刺激后 8hT细胞中CD69+、BrdU+双阳性细胞比例最高 (82 3 %± 7 2 % ) ,IFN γ+、IL 4+细胞百分率分别为 2 5 2 %± 3 7%和 3 4%± 1 6 % ,均显著高于未刺激组 (P <0 0 1 )。结论 :应用流式细胞术进行单个核细胞的增殖分析可以同时检测细胞表面活化抗原的表达、DNA合成及胞内细胞因子的分泌。此方法能够分析不同亚群细胞对于不同刺激原的反应和激活表型。敏感性高、重复性好 。Objective:To establish a new method for measuring lymphocytic proliferation effects and intracellular cytokines production by new Flew cyto metry assays.Methods:Periphral lymphocytes of 10 donors were co cultured with PMA(20 ng/ml) or PHA(20 μg/ml) for 2,4,6,8,10 hours respectively, then Brdu was added.T lymphocytic proliferation including CD69 expression,DNA synthesis and intracellular cytokines producing were assessed by 3 color flow cytometry.The optimal experimental condition were selected to improve the sensitivity of assays.Results:The optimal stimulation,were PMA 20 ng/ml,cell density 1×10 6/ml.the proper time of cell culturing was 8 h,with this condition the perontage of CD69 +?BrDU + bi expressin and IFN γ +?IL 4 + cells in peripheral lymphocytes were 82.3±7.2%, and 25.2±3.7%,3.4±1.6% respectively,which were significantly higher than those of control group( P <0.01) production.Conclusion:This method could determin the lymphocytic proliferation,phenotype analysis and cytokine production of the individual cells at single cell level.It may be well used to analyse the mononuclear cells proliferation effects in varies field.

关 键 词：流式细胞术 细胞增殖 细胞因子 

分 类 号：R392[医药卫生—免疫学]