Objective To establish a method of detecting different interleukin 22 ( IL-22 ) producing lymphocyte subpopulations in human peripheral blood by flow cytometry ( FCM ), and investigate its application in patients with tuberculosis ( TB ) and realize the role of IL-22 producing lymphocyte subpopiilations in anti -infection immunity against TB. Methods Peripheral blood samples collected from 22 TB patients and 18 healthy controls were mixed with RPMI-1640 culture medium at the scale of the same volume. After adding phorbol ester ( PMA ) and calcium ionomycin mixed for 2 h cultnring on the conditions of 37 ℃ and 5% CO2 ,monensin was added and continued being cultured for 4 h. The cells were collected and stained surface molecular or intracellular with flnorochrome-conjugated monoclonal antibodies. The respective proportions of different IL-22 and IL-17 producing lymphocyte subpopiilations were detected by FCM. Results The condition that the concentration of PMA was 1 OOng/mL and that of ionomycin was 1 μg/mL with 2 h being stimulated and cultured could produce IL-22 effectively. The proportions of IL-22 producing gamma delta T cells in TB patients (1.31% ) were obviously higher than those in healthy controls ( 1. 81 % ,P <0. 05 ). No statistical significance was found between the proportions of IL-22 producing positive cells in CD4 +T,CD8 + T, natural killer ( NK ) cells and B cells in TB patients and those in healthy controls ( P > 0. 05 ). No statistical significance was found between the proportions of Th22 and Thl7 in TB patients and those in healthy controls ( P>0.05 ). Conclusions The established easy, complete and reliable method for the detection of IL-22 producing lymphocyte subpopiilations and IL-22 producing gamma delta T rolls could art immunologiral effects in anti-infection immunity against TB.%目的 建立用流式细胞术(FCM)检测人外周血产白细胞介素22(IL-22)的不同淋巴细胞亚群的方法,并探讨其在结核病(TB)患者中的应用,以了解产IL-22的淋巴细胞亚群在抗结核感染免疫中的作用.方法 采集22例TB患者和18名健康对照者外周血,与相同体积RPMI-1640培养液混匀,加入佛波醇酯(PMA)和钙离子霉素,混匀后于37 ℃ 5% CO2静置培养2 h,再加入莫能霉素后继续培养4 h,收集细胞,用荧光素标记进行细胞表面抗原和胞内细胞因子染色,用FCM检测表达IL-22、IL-17的不同淋巴细胞亚群比例.结果 PMA浓度为100 ng/mL,钙离子霉素浓度为1 μg/mL,刺激培养2 h可最有效刺激IL-22的产生;TB患者外周血中产IL-22的γδT细胞比例(7.37%)明显高于正常人(1.81%,P<0.05),而在CD4+T、CD8+T、自然杀伤(NK)细胞和B细胞等其他细胞亚群中,产IL-22的阳性细胞比例在TB患者和健康对照者之间差异无统计学意义(P>0.05);Th22和Th17细胞比例在TB患者和健康对照者之间差异无统计学意义(P>0.05).结论建立了一套简便、完整、可靠的FCM检测产IL-22的淋巴细胞亚群的方法,产IL-22的γδT细胞在抗结核感染免疫中可能发挥免疫效应.
展开▼
