CAJ | 학술논문

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.
목적 탐토특응성피염(AD)환자외주혈T세포린산기순3격매(PI3K)신호전도통로활화정황급기림상의의.방법 검측38례AD환자적T세포PI3K통로활성,건강대조조38례.외주혈T림파세포분리채용Rosettsep T세포제취시제합제취,PI3K활성채용면역침정화매련면역흡부시험(ELISA),Akt급기린산화단백채용면역인적법,T세포증식채용새서람(MTT),백세포개소(IL)6화IL-10수평채용ELISA검측.결과 신선분리적AD환자외주혈T세포PI3K화Akt활성균현저고우건강대조조(P＜0.05).AD환자T세포재체외배양24 h후PI3K화Akt활성여건강대조조비교,차이무통계학의의(P＞0.05),건강대조조T세포용AD환자혈청부육24 h후PI3K화Akt활성현저증고(P＜0.05).PI3K특이성억제제LY294002현저억제AD환자T세포증식[(123±25)％화(195±28)％,P＜0.05]급분비IL-6화IL-10분별위[(431±64) ng/L화(823±128) ng/L,P＜ 0.05],[(120±21) ng/L화(213±35) ng/L,P＜0.05].신선분리적AD환자외주혈T세포PI3K화Akt활성여질병엄중정도지수(EASI)무상관.결론 AD환자외주혈T세포존재린산기순3격매신호전도통로활화이상,병여T세포증식화세포인자분비유관,제시AD환자외주혈중가능존재격활해통로적혈청인자.
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.