佛波酯对人外周血淋巴细胞CD3和CD4及CD8分子表达的作用

Regulation of CD3,CD4 and CD8 expressions on PMA-activated human peripheral T cells

目的 :分析佛波酯 (PMA)对细胞表面 CD3、CD4和 CD8表达的影响 ,及与胞浆内细胞因子分泌的关系。方法 :取 10例健康儿童及 2 7例呼吸道感染患儿外周血单个核细胞作体外培养后 ,用不同浓度及时间 PMA刺激后作胞浆内细胞因子及细胞表面分子标记物分析。结果 :与未刺激组相比 ,PMA不同浓度 (5、10、2 0和 5 0 ng/ ml)刺激4 h后 CD4表达均有明显下降 ,分别从 4 1.0 3%、4 0 .18%、4 2 .2 9%及 4 2 .85 %下调至 2 8.2 2 %、2 4 .4 4%、2 6 .0 9%及2 5 .70 % ,10 ng/ ml浓度刺激 4、6 h后细胞表面 CD4也有显著下调 ,分别从 4 5 .5 4%及 4 0 .5 6 %下调至 2 8.88%及2 0 .0 3% ,且 CD4表达与胞浆内细胞因子分泌呈负相关 (r=- 0 .6 0 1,P<0 .0 0 1)。PMA对 CD3(刺激前后分别为4 9.5 7%及 5 1.5 4% )及 CD8(刺激前后分别为 13.0 2 %及 12 .85 % )表达差异无显著性 ,而在 CD3+ T淋巴细胞中 ,CD4 -CD8-细胞仅占 5 .5 2 %。结论 :PMA刺激对辅助性 T淋巴细胞 (Th)表面 CD4表达有显著下调作用 ,且 Th细胞胞浆内细胞因子分泌越多 ,CD4分子下调越明显 ,而 PMA对 CD3及 CD8无明显下调作用。可采用 CD3+ CD8-表面标记的方法间接定义 Th细胞 ,替代 CD4分子 ,以避免 PMA这种效应对 Th1/ Th2数量检测所带来的困难。

Objective: To investigate the effect of mitogen Phorbol 12-myristate 13-Acetate (PMA) on CD3,CD4 and CD8 expression of human T-lymphocytes. Methods: Peripheral blood mononuclear cells from 37 blood samples stimulated in vitro with PMA at different concentrations (2,5,10,20 and 50 ng/ml for 4 hours) and time (10 ng/ml for 2,4 and 6 hours) were analyzed by 4-color flow cytometry (FCM). Results: Under different PMA stimulation protocols,significant CD4 down-regulation was observed,which was negatively correlated with intracellular cytokine secretion (r=-0.601,P<0.001),except for PMA stimulation at 10 ng/ml for 2-hours which showed no significant intracellular cytokine secretion. The expressions of CD3 and CD8 molecules after PMA activation were not significantly affected as compared with pre-activation. Among CD3 positive T-lymphocytes, CD4/CD8 double-negative cells only account for 5.52%. Conclusion: PMA has a significant down-regulation effect on CD4 molecules of Th cells,without altering the CD3 and CD8 expression. For quantitative analysis of Th1/Th2 variation,indirect method such as CD3+CD8- T cells can be used to define CD4+ Th cells stimulated by PMA in the future.