流式CBA法检测哮喘患儿Th1/Th2和Treg/Th17细胞因子失衡情况

Detection of Th1/Th2 and Treg/Th17 cytokines imbalance in children with asthma by flow cytometry CBA method

目的：哮喘是严重威胁儿童身心健康的慢性气道炎症性疾病,但目前哮喘发病的免疫机制尚未完全阐明.本研究采用流式CBA法检测哮喘患儿急性期血清中Th1、Th2、Th17和Treg类细胞因子的表达情况,从而探讨在儿童哮喘发病过程中Th1/Th2以及Treg/Th17失衡情况.方法：以2013.10-2014.12于山东大学齐鲁儿童医院初次确诊哮喘或停止规范吸入激素治疗大于3个月后哮喘复发的患儿（哮喘组,n=81）和同期健康体检儿童（对照组,n=38）为研究对象.收集两组患儿外周血,采用流式细胞术检测外周血中T淋巴细胞亚群的水平,并结合流式CBA法检测血清中Th1、Th2、Treg和Th17细胞因子IL-2、IL-4、IL-6、IL-10、TNF、IFN-γ、IL-17A以及IL-12/IL-23p40的表达情况.结果：①一般资料统计分析,两组在年龄和性别组成上没有显著性差异;②与对照组比较,各年龄组CD3^＋T细胞、CD4^＋T细胞比例及2岁以内CD4/CD8均明显降低（P〈0.01或P〈0.05）,各年龄组的CD8^＋T细胞比例及大于2岁的CD4/CD8在两组无明显差别（P〉0.05）;③与对照组比较,哮喘组患儿血清中Th1类细胞因子IL-2、IFN-γ含量显著增高,具统计学意义（P〈0.05）,而两组患儿血清中TNF含量没有明显差异（P〉0.05）;④哮喘组患儿血清Th2类细胞因子IL-4、IL-6比对照组低,有统计学意义（P〈0.01或P〈0.05）;而IL-10浓度则明显高于对照组（P〈0.05）;⑤哮喘组患儿血清Treg细胞因子IL-12/IL-23p40显著低于对照组（P〈0.01）,而Th17细胞因子IL-17A的表达在两组之间没有显著差异.结论：儿童哮喘发病过程中Th1细胞功能亢进,Th2和Treg细胞功能降低,Th1/Th2和Treg/Th17细胞因子失衡状态可能是哮喘发病的重要原因,流式CBA法能快速、 准确检测Th1/Th2和Treg/Th17细胞因子的表达.

Objective： Asthma is a chronic airway inflammatory disease which seriously threatens the physical and mental health of children. In this study, the expression of Th1, Th2, Th17 and Treg cytokines in the serum of children with asthma during the acute phase was detected by flow cytometry CBA, so as to explore the imbalance of Th1/Th2 and Treg/Th17 in children with asthma. Methods： Eighty-one children with asthma （asthma group） and 38 healthy children （control group） were recruited in this study, who were admitted to the Qilu Children＇ Hospital of Shandong University from October 2013 to December 2014. All asthmatic children were diagnosed as asthma or stop inhaled hormone therapy more than 3 months after the recurrence. Collection of both groups of children with peripheral blood, the levels ofT-cells subgroups were determined by flow cytometry. The expression of peripheral IL-17A and IL-12/IL-23p40 of all subjects was measured using CBA method. Combined with the flow cytometry assay for the detection of serum CBA in Thl, Th2, Treg and IL-4, Th17 cytokines IL-2, IL-6, IL-10, TNF, IFN-γ, IL-17A and IL-12/IL-23p40 expression condition. Results： 1.There were no differences in age, sex between both groups; 2. Compared with the control group, each age group the proportion of cells in CD4^＋ and CD3^＋T, 2 years old CD4/CD8 were significandy lower （P〈0.01 or P〈0.05） , the proportion of CD8^＋T cells in various age groups and more than 2 year old CD4/CD8 in both groups had no significant difference （P〉0.05） ; 3. Compared with the control group, serum Thl cytokines in children with asthma IL-2 and IFN-~/were significantly increased （P〈0.05） , and the content of TNF in serum of the two groups had no significant difference （P〉0.05） ; 4. There was statistically significant that asthma group serum Th2 cytokine IL-4, IL-6 lower than the control group （P〈0.01 or P〈0.05） ; while the IL-10 concentration is higher than control group （P〈0.05） ; 5. The asthma patients serum Treg cytokine IL-12/IL-23p40 was significantly lower than control （P〈0.01） , and there was no significant difference between the two groups in the expression of Th17 cytokine IL-17A. Conclusion： Thl cell hyperfunction, the function of Th2 and Treg cells decreased, and Th1/Th2 and Treg/Th17 cytokine imbalance may play an important role in the pathogenesis of asthma with children. And the CBA method can be used to exactly evaluate the expression of Th1/Th2 and Treg/Th17 cytokines.