他克莫司及金黄色葡萄球菌肠毒素B对特应性皮炎外周血Th17细胞的影响

【摘要】 目的 探讨Th17细胞在急性期特应性皮炎（AD）患儿外周血单一核细胞（PBMC）中的表达，他克莫司和金黄色葡萄球菌肠毒素B（SEB）对AD患儿外周血Th17细胞的影响。 方法 分离急性期中、重度AD患儿及健康对照儿童PBMC，分别加入佛波酯和离子霉素、他克莫司、金黄色葡萄球菌肠毒素B培养，流式细胞仪检测Th17、Th1、Th2细胞比例，ELISA检测培养上清中IL-17、IFN-γ、IL-4表达，RT-PCR法检测Th17特异性核转录因子RORγt mRNA表达。 结果 佛波酯和离子霉素处理后，AD患儿外周血Th17、Th2细胞比例及相关细胞因子IL-17、IL-4水平明显高于健康对照组（P < 0.01）。Th1细胞比例及IFN-γ水平低于健康对照组（P < 0.01），ROR γt mRNA高于健康对照组（P < 0.01）。他克莫司处理后，AD组和对照组IL-17、IFN-γ、IL-4及ROR γt mRNA水平均显著降低（P < 0.01）。SEB处理后，AD组 Th17细胞比例，IL-17和 RORγt mRNA水平显著高于对照组（P < 0.01），Th1细胞比例及IFN-γ水平低于对照组（P < 0.01），Th2细胞比例及IL-4水平高于对照组（P < 0.01）。 结论 他克莫司对AD患儿和健康对照PBMC分泌IL-17、IFN-γ、IL-4均有明显的抑制作用。SEB增强AD患儿外周血Th17细胞表达。

Effect of tacrolimus and Staphylococcus aureus enterotoxin B on T helper 17 cells in peripheral blood from patients with atopic dermatitis

Yu Lu, Wang Hua, Luo Xiaoyan. Department of Dermatology, Children's Hospital, Chongqing Medical University, Chongqing 400014, China Corresponding author: Luo Xiaoyan, Email: luoxycq@hotmail.com 【Abstract】 Objective To determine the proportion of T helper 17（Th17） cells in peripheral blood mononuclear cells （PBMCs） from children with acute atopic dermatitis （AD）, and to evaluate the effect of tacrolimus and Staphylococcus aureus enterotoxin B （SEB） on the proportion. Methods PBMCs were separated from 86 children with moderate to severe acute AD and 81 healthy children, and cultured in the presence of phorbol ester （PMA） and ionomycin with or without tacrolimus or SEB for different durations. Then, flow cytometry was performed to determine the proportions of Th17, Th1 and Th2 cells, enzyme-linked immunosorbent assay （ELISA） to measure the levels of interleukin （IL）-17, interferon （IFN）-γ and IL-4 in the culture supernatant of these cells, and real-time fluorescence-based quantitative PCR（RT-PCR） to detect the expression level of RORγt mRNA. Statistical analysis was done by using two independent samples t-test, chi-square test and analysis of variance. Results After stimulation with PMA and ionomycin, there was a significant increase in the proportions of Th2 and Th17 cells in, supernatant levels of IL-17 and IL-4 and mRNA expression level of RORγt of, but a significant decrease in Th1 cell percentage in and IFN-γ supernatant level of, PBMCs from patients with AD compared with PBMCs from the healthy controls （all P < 0.01）. The combination treatment with tacrolimus significantly reduced the supernatant levels of IL-17, IFN-γ, IL-4 and RORγt mRNA expression level of PBMCs from both the patients and healthy controls compared with those treated with PMA and ionomycin only （all P < 0.01）. In comparison with the healthy controls, the patients with AD showed significantly higher proportions of Th17 and Th2 cells in, supernatant levels of IL-17 and IL-4 of, and expression level of RORγt mRNA of, but significantly lower proportion of Th1 cells in and IFN-γ supernatant level of, PBMCs after SEB treatment （all P < 0.01）. Conclusions Tacrolimus evidently inhibits the secretion of IL-17, IFN-γ and IL-4 by PBMCs from both children with AD and healthy children, while SEB can up-regulate the proportion of peripheral blood Th17 cells in children with AD.