特应性皮炎不同T细胞亚群平衡紊乱与中医证型关联及YAP对其调控作用的研究

Relationship Between Imbalance of Different T Cell Subsets in Atopic Dermatitis and Traditional Chinese Medicine Syndrome Types and the Regulation of YAP on It

目的研究辅助性T细胞(Th)1/Th2/Th17/调节性T细胞(Treg)比例在特应性皮炎(AD)不同中医证型之间的差异,并探索Yes相关蛋白(YAP)在不同T细胞亚群中的表达量与其平衡紊乱之间的关联。方法收集AD患者35例(其中心火脾虚证21例、脾虚蕴湿证9例、血虚风燥证5例)及健康对照者24例,分析其临床特征,流式细胞仪检测不同组Th1/Th2/Th17/Treg细胞比例,酶联免疫吸附测定(ELISA)法检测细胞因子的表达,分离外周血单个核细胞(PBMC)并诱导分化为不同T细胞亚群后检测YAP mRNA的表达量。结果AD组及健康对照组在年龄、性别上比较差异无统计学意义,AD患者中心火脾虚组AD严重程度评分(SCORAD)高于脾虚湿蕴组和血虚风燥组(P<0.001),性别、年龄、病程、嗜酸性粒细胞计数、IgE差异无统计学意义;Th2及Th17比例在心火脾虚组升高(P<0.05),Treg在心火脾虚组降低(P<0.05)、在血虚风燥组升高(P<0.05),Th1/Th2及Th17/Treg比例在心火脾虚组均升高(P<0.05);Th1相关细胞因子干扰素(IFN)-γ在心火脾虚组降低(P<0.01),Th2相关细胞因子白细胞介素(IL)-4在心火脾虚组升高(P<0.001),Th17相关细胞因子IL-17A及IL-21在心火脾虚组升高(P<0.01),Treg相关细胞因子IL-10在心火脾虚组降低(P<0.001),其余IL-6、转化生长因子(TGF)-β及IL-2均无明显变化;健康对照组中YAP mRNA在Treg中明显升高,(P<0.05),而在AD组中YAP mRNA在Th17中表达升高,在Treg中相对降低,YAP mRNA在Th17中的表达在心火脾虚组明显升高(P<0.01),在Treg中的表达在心火脾虚组明显降低(P<0.05),在Th1及Th2中的表达差异无统计学意义。结论在AD中存在Th2/Th1、Th17/Treg平衡紊乱,且在心火脾虚证中Treg下降、Th2及Th17为主导的免疫反应更明显,YAP在Treg中的表达下调、Th17中的表达上调很可能是介导AD Th17/Treg平衡向Th17偏移的关键因素,且这一特征在心火脾虚证中更明显。

Objective In order to study the differences of Th1/Th2/Th17/Treg ratio in different traditional Chinese medicine(TCM)syndromes of atopic dermatitis(AD),and to explore the relationship between the expression of Yes-associated protein(YAP)in different T cell subsets and its imbalance.Methods Thirty-five cases of AD patients(including 21 cases of heart fire and spleen deficiency syndrome,9 cases of spleen deficiency and dampness accumulation syndrome,5 cases of blood deficiency and wind dryness syndrome)and 24 healthy controls were collected.The ratio of Th1/Th2/Th17/Treg cells in different groups was detected by flow cytometry,the expression of cytokines was detected by enzyme-linked immunosorbent assay(ELISA),and the expression of YAP mRNA was measured after peripheral blood mononuclear cells were isolated and induced to differentiate into different T cell subsets.Results The scoring atopic dermatitis(SCORAD)index of AD patients with heart fire and spleen deficiency group was higher than that of spleen deficiency and dampness accumulation group and blood deficiency and wind dryness group(P<0.001),but gender,age,course of disease,eosinophil count and IgE had no statistical significance.The proportion of Th2 and Th17 increased(P<0.05)while Treg decreased in the heart fire and spleen deficiency group.And the proportion of Treg increased in blood deficiency and wind dryness group(P<0.05).Th1/Th2 and Th17/Treg ratio increased in the heart fire and spleen deficiency group(P<0.05).Th1 related cytokines IFN-γdecreased(P<0.01)in the heart fire and spleen deficiency group.While Th2 related cytokines IL-4 increased(P<0.001).Th17 related cytokines IL-17A and IL-21 increased(P<0.01).Treg related cytokines IL-10 decreased(P<0.01).IL-6,TGF-βand IL-2 had no significant changes.In healthy control group,YAP mRNA was significantly increased in Treg cells(P<0.05),while in AD group,YAP mRNA expression was increased in Th17 and decreased in Treg.The expression of YAP mRNA was significantly increased in Th17(P<0.01)and decreased in Treg(P<0.05)in heart fire and spleen deficiency group,but there were no statistical significances in Th1 and Th2.Conclusion There is imbalance of Th2/Th1 and Th17/Treg in AD,and the decreasing of Treg cells and the immune response dominated by Th2 and Th17 are more obvious in heart fire and spleen deficiency syndrome.The down-regulation of YAP expression in Treg cells and the up-regulation of YAP expression in Th17 cells of AD may be the key factors that mediate the shift of Th17/Treg balance to the side of Th17 in AD,and this feature is more obvious in heart fire and spleen deficiency syndrome.