流式细胞术检测γδΤ细胞内白细胞介素-2的临床应用价值

The clinical application of flow cytometry for detecting intracellular IL-2 in γδΤ cells

目的建立一种人外周血γδΤ细胞细胞内白细胞介素-2(IL-2)的检测方法。方法分离获取健康人外周血单个核细胞(PBMC),用结核杆菌低分子多肽抗原(Mtb-Ag)刺激PBMC,获取较高比例的γδΤ细胞。用佛波醇酯+离子霉素(PMA+IM)刺激PBMC和γδΤ细胞,用蛋白转运抑制剂(Brefeldin A,BFA)阻断细胞内因子的分泌,用皂角素破膜,流式细胞术检测CD3+T细胞内CD69阳性率,以确定最佳的破膜时间,用流式细胞术检测γδΤ细胞内的IL-2的表达。不同组间CD3+T细胞内CD69阳性率及细胞内IL-2的阳性率比较采用单因素方差分析,组间两两比较采用最小显著性(LSD)法检验.。结果刺激时间不同,CD3+T细胞胞内CD69的阳性率表达不同,皂角素破膜时间为15 min时,CD3+T细胞胞内CD69的阳性率最高,达(87.82±2.28)%,与处理5 min[CD69的阳性率(66.86±1.99)%]和25 min[CD69的阳性率(81.73±2.51)%]比较,差异有统计学意义(F=112.81,P<0.05);不同处理组γδΤ细胞内IL-2表达不同,γδΤ细胞受PMA和IM刺激并有BFA存在时表达最高,达(50.65±6.25)%,与γδΤ细胞组(0.55±0.07)%、γδΤ细胞+PMA+IM组(0.91±0.12)%和PBMC+PMA+IM+BFA组(0.21±0.03)%比较,差异有统计学意义(F=321.07,P<0.05)。结论流式细胞术是一种检测γδΤ细胞内IL-2的较好方法,为γδΤ细胞内其他分子的检测分析奠定了方法学基础。

Objective To establish a method for detecting intracellular IL-2 in human peripheral blood γδΤ cells.Methods Healthy human peripheral blood mononuclear cells （PBMCs） were isolated,and then stimulated with low molecular peptide antigen of mycobacterium tuberculosis （ Mtb-Ag ） . A high proportion of γδΤ cells were obtained. PBMCs and γδΤ cells were excited with phorbol myristate acetate （PMA） and ionomycin（IM）.Protein transport inhibitor （Brefeldin A,BFA） was used to block the secretion of cytokines and saponin was taken for membrane permeabilization.Flow cytometry was applied to detect the expression of CD69 in CD3＋ T cells to explore the appropriate time of membrane permeabilization.Then flow cytometry was implemented to detect the expression of intracellular IL-2 in γδΤ cells. Analysis of Variance （ ANOVA） and Least Significant Difference（ LSD） test were conducted to determine the statistical difference. Results CD3＋ T cells with 15 minutes of membrane permeabilization had the highest expression of CD69 （87.82±2.28）% compared with those with 5 minutes and 25 minutes （F = 112.81,P〈0.05）.γδΤcells with the stimulation of PMA,IM and blocker of BFA （γδΤ＋PMA＋IM＋BFA） had the highest expression of intracellular IL-2 （50.65±6.25）% compared with γδΤ group,γδΤ＋PMA＋IM group and PBMC＋PMA＋IM＋BFA group（F = 321.07,P〈0.05）.Conclusion Flow cytometry is an appropriate method for the detection＆amp;nbsp;of intracellular IL-2 in γδΤ cells,and it may be applicable to detecting other cytokines in γδΤ cells.