【摘要】 目的:研究急性移植物抗宿主病(acute graft-versus-host disease,a GVHD)患者CD4+T细胞中STAT3基因启动子DNA病理性低甲基化的分子调控机制。方法:收集行同胞全相合异基因造血干细胞移植的42例患者的血液样本。采用实时定量PCR和Western印迹检测各组患者生长阻滞与DNA损伤诱导蛋白45A(growth arrest and DNA damage-inducible protein 45 alpha,GADD45A)的表达水平,免疫共沉淀检测各组患者高迁移率族蛋白1(high mobility group box-1 protein,HMGB1)与GADD45A的结合水平,染色质免疫共沉淀检测各组患者HMGB1和GADD45A与STAT3启动子的结合水平;正常CD4+T细胞中过表达HMGB1及干扰GADD45A表达后用Western印迹检测STAT3表达水平,亚硫酸盐测序检测STAT3启动子区DNA甲基化水平。结果:与未发生a GVHD患者比较,a GVHD患者外周血CD4+T细胞中GADD45A表达水平明显升高;HMGB1与GADD45A的结合显著增加;HMGB1,GADD45A与STAT3启动子的结合水平均明显升高,且STAT3启动子区HMGB1,GADD45A结合水平与其DNA甲基化水平呈高度负相关(分别r=0.719,r=0.840;P<0.01)。与单纯过表达HMGB1相比,正常CD4+T细胞中过表达HMGB1叠加干扰GADD45A表达后STAT3表达明显降低,STAT3启动子DNA甲基化水平明显升高。结论:CD4+T细胞中HMGB1和GADD45A表达升高是异基因造血干细胞移植后患者STAT3启动子DNA低甲基化,STAT3高表达,进而诱发a GVHD的重要因素。更多还原

【Abstract】 Objective: To study the molecular mechanism for DNA hypomethylation of STAT3 promoter in CD4+ T cells from acute graft-versus-host disease(aGVHD) patients. Methods: We collected CD4+ T cells from peripheral blood of 42 patients who underwent allogeneic hematopoietic stem cell transplantation(allo-HSCT) from HLA-identical sibling donors. GADD45 A expression level in CD4+ T cells was measured by real-time PCR and Western blot. The binding level between HMGB1 and GADD45 A in CD4+ T cells was analyzed by coimmunoprecipitation, while the binding levels of HMGB1/GADD45 A with STAT3 promoter were detected by chromatin immunoprecipitation-quantitative real-time PCR(ChIP-qPCR). After overexpression of HMGB1 and knockdown of GADD45 A in normal CD4+ T cells, STAT3 expression and DNA methylation were measured by Western blot and bisulfite sequencing PCR, respectively. Results: GADD45 A expression was significantly up-regulated in patients with aGVHD compared with that in the patients without aGVHD. More HMGB1-GADD45 A complexes were found in CD4+ T cells from patients with aGVHD compared with that in patients without aGVHD. The bindings of HMGB1/GADD45 A with STAT3 promoter were significantly increased, and the binding levels of HMGB1/GADD45 A were negatively correlated with STAT3 promoter DNA methylation. The expression of STAT3 was significantly reduced and the DNA methylation of STAT3 promoter was significantly increased in CD4+ T cells with overexpression of HMGB1 and knockdown of GADD45 A compared with CD4+ T cells only with overexpression of HMGB1. Conclusion: The increased expression of HMGB1/GADD45 A plays an importent role in STAT3 promoter DNA hypomethylation, thereby promoting STAT3 expression in CD4+ T cells from a GVHD patients.更多还原