系统性红斑狼疮患者外周血T细胞磷酸肌醇3激酶信号通路异常活化
Abnormal signaling activity of phosphatidylinositol 3-kinase pathway in peripheral blood T cells from patients with systemic lupus erythematosus
目的 研究系统性红斑狼疮(SLE)患者外周血T细胞中磷酸肌醇3激酶(PI3K)信号通路活化情况及其临床意义.方法 检测28例SLE患者的T细胞PI3K通路活性.8例类风湿关节炎患者为对照,另以15名健康者为正常对照组.外周血T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ELISA法,Akt及其磷酸化蛋白运用免疫印迹法,T细胞增殖采用MTT法,细胞因子水平采用ELISA检测.结果 新鲜分离的SLE患者外周血T细胞PI3K和Akt活性均显著高于正常对照组和类风湿关节炎组;SLE患者T细胞在体外培养24 h后PI3K和Akt活性与正常对照组差异无统计学意义,而正常对照组T细胞用SLE患者血清孵育24 h后PI3K和Akt活性显著增高;PI3K特异性抑制剂LY294002显著抑制SLE患者T细胞增殖(125±21 vs 197±26,P<0.05)及分泌白细胞介素(IL)-6和IL-10[分别为(425±69)ng/L vs(816±116)ng/L,P<0.05;(114±18)ng/L vs(207±31)ng/L,P<0.05].新鲜分离的SLE患者外周血T细胞PI3K和Akt活性与SLEDAI无相关关系.结论 SLE患者T细胞存在PDK信号通路异常活化,并与T细胞增殖和细胞因子分泌有关,SLE外周血中可能存在激活该通路的血清因子.
更多Objective To investigate the activity of phasphatidylinositol 3-kinase (PI3K) signal pathway,a cytoplasmic signaling pathway known to play an important role in T cell activation,in peripheral blood T cells from systemic lupus erythematosus (SLE) patients.Methods T cells were isolated from the peripheral blood samples of 28 SLE patients,5 males and 23 females,with RosettSep T cell purification kit.PI3K activity was determined by immunoprecipitation and ELISA,and Western blotting was used to measure the Akt and phosphorylated Akt protein expression.T cell proliferation and cytokine production was examined by MTT test and ELISA respectively.Fifteen healthy adults and 8 active rheumatoid arthritis patients were used as controls.The T cells from the SLE patients and normal controls were treated with 10% normal control serum of SLE serum for 24 h ( "rest" ) and then to detect the PI3K and Akt activity.Some T cells from the SLE patients were stimulated with CD3/CD28 mono-antibodies or CD3/CD28 monoantibodies + LY294002 ,a specific PI3K inhibitor,and then the proliferation and secretion of IL-6 and IL-10 were analyzed.Results Compared with the healthy controls and rheumatoid arthritis patients,the activity levels of PI3K and Akt in the T cells of peripheral blood from the SLE patients were significantly increased.T cells allowed to " rest" for 24 hours in culture medium showed a reversal of the changes in activity of PI3K and Akt.The activity of PI3K pathway was increased in the T cells from healthy controls when cultured with SLE serum.The proliferation and IL-6 and IL-10 secretion of the T cells from SLE patients cultured with LY294002 were inhibited.The PI3K and Akt activity levels of the T cells from SLE patients were not related to SLE disease activity index (SLEDAI).Conclusion The T cells from SLE patients show an abnormal activation of PI3K pathway which may be due,at least in part,to their exposure to relevant serum factors.
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