特应性皮炎患者外周血T细胞磷酸肌醇3激酶信号转导通路的表达

Expression of the phosphatidylinositol 3-kinase signaling pathway in peripheral blood T cells from patients with atopic dermatitis

目的 探讨特应性皮炎（AD）患者外周血T细胞磷酸肌醇3激酶（PI3K）信号转导通路活化情况及其临床意义.方法 检测38例AD患者的T细胞PI3K通路活性,健康对照组38例.外周血T淋巴细胞分离采用Rosettsep T细胞提取试剂盒提取,PI3K活性采用免疫沉淀和酶联免疫吸附试验（ELISA）,Akt及其磷酸化蛋白采用免疫印迹法,T细胞增殖采用噻唑蓝（MTT）,白细胞介素（IL）6和IL-10水平采用ELISA检测.结果 新鲜分离的AD患者外周血T细胞PI3K和Akt活性均显著高于健康对照组（P＜0.05）.AD患者T细胞在体外培养24 h后PI3K和Akt活性与健康对照组比较,差异无统计学意义（P＞0.05）,健康对照组T细胞用AD患者血清孵育24 h后PI3K和Akt活性显著增高（P＜0.05）.PI3K特异性抑制剂LY294002显著抑制AD患者T细胞增殖[（123±25）％和（195±28）％,P＜0.05]及分泌IL-6和IL-10分别为[（431±64） ng/L和（823±128） ng/L,P＜ 0.05],[（120±21） ng/L和（213±35） ng/L,P＜0.05].新鲜分离的AD患者外周血T细胞PI3K和Akt活性与疾病严重程度指数（EASI）无相关.结论 AD患者外周血T细胞存在磷酸肌醇3激酶信号转导通路活化异常,并与T细胞增殖和细胞因子分泌有关,提示AD患者外周血中可能存在激活该通路的血清因子.

Objective To estimate the activity of the phosphatidylinositol3-kinase （PI3K） signaling pathway in peripheral blood T cells from patients with atopic dermatitis （AD）,and to investigate its clinical significance.Methods T cells were isolated by using the Rosettsep T cell purification kit from the peripheral blood of 38 patients with AD and 38 healthy human controls,and classified into several groups to be treated with anti-CD3 monoclonal antibody,anti-CD28 monoclonal antibody,and LY294002 （an inhibitor of PI3K） respectively.The activity of PI3K signaling pathway in T cells was estimated by immunoprecipitation and enzyme-linked immunosorbent assay （ELISA）.Western blot was performed to measure the expressions of total Akt and phosphorylated Akt in T cells,methyl thiazolyl tetrazolium （MTT） assay to examine the proliferation of T cells,and ELISA to determine the levels of interleukin 6 （IL-6） and IL-10.Results The activity of PI3K and Akt was significantly higher in freshly isolated patient-derived T cells than in control-derived T cells （both P 〈 0.05）.However,the difference in the activity of PI3K and Akt between patient-derived and control-derived T cells disappeared （both P 〉 0.05） after 24-hour in vitro culture.The activity of PI3K and Akt in control-derived T cells was significantly increased after 24-hour incubation with sera from the patients with AD （both P 〈 0.05）.In addition,compared with patient-derived T cells treated with patients＇ sera or anti-CD3/CD28 monoclonal antibody alone,those treated with the combination of LY294002 and patients＇ sera or anti-CD3/CD28 monoclonal antibody showed a significant decrease in the proliferative activity （63% ± 11% vs.123% ± 25%,125% ± 22% vs.195% ± 28%,both P〈 0.05）,supematant levels of IL-6 （（168 ± 33） vs.（265 ± 46） ng/L,（431 ± 64） vs.（823 ± 128） ng/L,both P〈 0.05） and IL-10 （（56 ± 14） vs.（98 ± 25） ng/L,（120 ± 21） vs.（213 ± 35） ng/L,both P〈 0.05）.Eczema area and severity index （EASI） was unassociated with the activity of PI3K or Akt in fresh T cells from patients with AD （both P 〉 0.05）.Conclusions The PI3K signaling pathway is abnormally activated in peripheral blood T cells from patients with AD,which is associated with the proliferation of,as well as secretion of cytokines by,T cells,suggesting that there exist serum factors activating this pathway in peripheral blood of patients with AD.