异基因造血干细胞移植患者外周血CD4+T细胞中STAT3启动子区DNA甲基化水平与急性移植物抗宿主病的关系

摘要

Objective:To study the relationship between acute graft versus host disease (aGVHD) and the methylation status of the STAT3 promoter in peripheral blood CD4+ T cells from patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT).Methods:We collected 40 patients who underwent allo-HSCT from HLA-identical sibling donors.Serum IL-10,TGF-β1,IL-17A and IL-17F levels were detected by ELISA.Foxp3 cytotoxic T-lymphocyte-associated protein 4 (CTLA4),IL-10,TG F-β 1,RO Rγt,IL-17A and IL-17F mRNA levels in CD4+ T cells were measured by real-time PCR.STAT3 expression levels were detected by real-time PCR and Western blot,and promoter DNA methylation was analyzed by bisulfite sequencing PCR (BSP).Results:IL-10 and TGF-β1 levels were significantly down-regulated,while IL-17A and IL-17F levels were significantly up-regulated in patients with aGVHD compared with patients without aGVHD.Foxp3,CTLA4,IL-10,TGF-β1 mRNA levels were significantly down-regulated,while RORγt,IL-17A,IL-17F mRNA levels were significantly up-regulated in patients with aGVHD compared with patients without aGVHD.STAT3 expression was increased,while STAT3 promoter DNA was hypomethylated in patients with aGVHD compared with those without aGVHD.The STAT3 mRNA level was negatively correlated with STAT3 promoter DNA methylation.Conclusion:The imbalance of Treg/fh17 in CD4+ T cells from patients after allo-HSCT is a key factor for triggering aGVHD,and the DNA hypomethylation of STAT3 promoter could promote its expression in CD4+ T cells and contribute to the imbalance.%目的:探讨异基因造血干细胞移植患者外周血CD4+T细胞中STAT3启动子区DNA甲基化水平与急性移植物抗宿主病(acute graft versus host disease,aGVHD)的关系.方法:收集行同胞全相合异基因造血干细胞移植的40例患者的血液样本,ELISA检测各组患者血清IL-10,TGF-β1,IL-17A,IL-17F等细胞因子水平;实时定量PCR检测各组患者外周血CD4+T细胞中Treg(Foxp3,CTLA4,IL-10,TGF-β1)和Th17(RORγt,IL-17A,IL-17F)相关基因的转录水平;实时定量PCR和Western印迹检测各组患者STAT3的表达水平;亚硫酸氢盐处理后测序(bisulfite sequencing PCR,BSP)法检测各组患者STAT3基因启动子区DNA甲基化水平.结果:与未发生aGVHD患者比较,aGVHD患者血清中IL-10及TGF-β1水平明显降低,IL-17A及IL-17F水平明显升高;aGVHD患者外周血CD4+T细胞中Foxp3,CTLA4,IL-10,TGF-β1转录水平明显降低,RORγt,IL-17A,IL-17F转录水平明显升高;aGVHD患者外周血CD4+T细胞中STAT3的表达水平明显升高,STAT3启动子区DNA甲基化水平明显降低,且STAT3表达水平与其启动子区DNA甲基化水平呈明显负相关.结论:Treg/Th 17的比例失衡是异基因造血干细胞移植后患者发生aGVHD的重要因素,STAT3启动子区DNA低甲基化可能介导STAT3的过度表达,参与Treg/Th17的比例失衡.