【摘要】 目的该研究通过探讨不同的刺激培养条件和检测方法对Th17细胞频率的影响,建立和优化流式细胞术检测人外周血Th17细胞频率实验方案。方法采用不同浓度的佛波酯(PMA,终浓度50、100、150、200、250、300ng/mL)与离子霉素(Ionomycin)1μg/mL对健康人外周血进行刺激培养,分别于刺激后4、6、8、10和12h用流式细胞术分析Th17细胞频率,并检测PMA刺激前后细胞表面与胞膜内CD4平均荧光强度(MFI)和CD4阳性T细胞比例。结果 PMA 150ng/m+Ionomycin 1μg/mL刺激全血6h后,Th17细胞频率高,刺激的效果较好(P<0.05)。刺激后检测细胞膜内CD4MFI低于先标记膜表面CD4后刺激组(P<0.05),Th17细胞频率和CD4阳性T细胞比例均高于后者,差异无统计学意义(P>0.05)。结论采用终浓度为PMA 150ng/mL+Ionomycin 1μg/mL刺激培养全血6h后,测定胞浆内CD4和IL-17水平是检测人Th17细胞频率简便有效的方法。更多还原

【Abstract】 Objective To investigate the effect of different stimulating culture conditions and detecting methods on the frequency of Th17 cells and to set up and optimize the experimental method for detecting the frequencies of human Th17 cells in peripheral blood by flow cytometry(FCM).Methods The peripheral blood in the healthy people was performed the stimulating culture by phorbol myristate acetate(PMA)at different final concentrations(50,100,150,200,250,300ng/mL)and ionomycin 1μg/mL.The frequencies of Th17 cells were assessed by FCM after different stimulating time points(4,6,8,10,12h).The mean fluorescence intensity(MFI)and percentage of the ratio of CD4 positive cells on the cellular surface and inside cell membrane were measured before and after PMA stimulation.Results The frequency of Th17 cells was highest with the good stimulation effect under the condition of PMA 150ng/mL and ionomycin1μg/mL stimulation for 6h(P<0.05).MFI of CD4 inside cellular membrane after stimulation was lower than that in the cells with stimulation after labeling membrane surface CD4(P<0.05),and the Th17 cell frequencies and the ratio of CD4 positive cells were higher than those in the latter,but the differences were not statistically significant(P>0.05).Conclusion After stimulating whole blood with the final concentration of PMA 150ng/mL and ionomycin1μg/mL for 6h,measuring intracytoplasm CD4 and IL-17 levels is the simple and effective method for detecting the frequency of human Th17 cells.更多还原