不同刺激剂和培养环境对CD4+、CD8+T细胞内细胞因子表达的影响

目的探讨不同的刺激剂和不同的培养条件对CD4 和CD8 T细胞内细胞因子表达的影响。方法分离正常人的外周血单个核细胞(PBMC),分别加入3种不同的刺激剂(PHA,抗CD3和抗CD28mAb,PMA和离子霉素),置于4种不同的培养环境下(室温,37℃水浴,37℃培养箱,37℃50mL/LCO2培养箱)培养4·5~5h。收集细胞,以荧光素-mAb标记后,用流式细胞术分析CD4 、CD8 T细胞内IL-2、IFN-γ和TNF-α的表达。结果CD4 和CD8 T细胞内细胞因子的表达,随着刺激剂的不同而有所差别,且在上述4种培养环境中,PMA的刺激效果最强,抗CD3mAb次之,PHA的刺激效应最弱。以上述3种刺激剂刺激后,不同培养条件对T细胞内细胞因子的表达有一定的影响。室温培养时几乎检测不到细胞因子的表达,而在37℃水浴、37℃培养箱和37℃50mL/LCO2培养箱中培养时,表达细胞因子的CD4 和CD8 T细胞的百分率差异无统计学意义(P>0.05)。结论不同刺激剂体外刺激T细胞表达细胞因子的效应不同,依次为PMA和离子霉素>抗CD3和抗CD28mAb>PHA。体外刺激培养的T细胞活化过程中,温度是重要的条件,CO2无明显影响。

Cytokine expression of CD4+ and CD8+ T cells induced by different stimuli under different culture conditions

AIM: To investigate the effect of different stimuli and different culture conditions on the cytokine expression of CD4+ and CD8+ T cells. METHODS: PBMCs were isolated from normal human peripheral blood and cultured with three kinds of stimuli (PHA, anti-CD3 and anti-CD28 mAbs, PMA and ionomycin) under four different culture conditions (room temperature, 37 degrees C water bath, 37 degrees C incubator, 37 degrees C and 50 mL/L CO2 incubator) for 4.5 ~ 5 h and intracellular cytokines (IL-2, IFN-gamma and TNF-alpha) in T cells were assessed by flow cytometry (FCM). RESULTS: Cytokine expression of CD4+ and CD8+ T cells varied when treated with different stimuli under different conditions. PMA had the strongest stimulative effect on cytokine expression of PBMC, while the effect of anti-CD3 mAb was weaker, and that of PHA was the weakest. Different culture conditions did not greatly change the cytokine expression profile of T cells (P>0.05) except that there were very few cytokine producing cells when PBMCs were cultured at room temperature. CONCLUSION: Anti-CD3 and anti-CD28 mAbs, PMA and ionomycin are recommended for the stimulation of PBMCs and detection of intracellular cytokine using FCM. Culture temperature but not the concentration of CO2 is the most important factor during the short term T cell stimulation.