PD-1基因启动子区域甲基化水平对慢性乙肝患者外周血单个核细胞上PD-1表达的影响

摘要

目的 探讨慢性乙型肝炎(CHB)患者外周血单个核细胞(PBMCs)上程序性死亡受体-1(PD-1)表达和PD-1启动子区域甲基化水平的差异及其影响因素,以及两者之间的关系.方法 筛选2009年9月至2011年9月在中南大学湘雅二医院传染科门诊就诊144例CHB患者和18名健康献血员,流式细胞术(FCM)检测PBMCs表面表达PD-1的细胞比例,同时进行肝功能检测；时间分辨荧光免疫分析法检测CHB患者血清HBV标志物水平,荧光定量PCR检测血清HBV DNA载量；亚硫酸氢钠处理PBMCs细胞DNA,PCR扩增PD-1启动子基因片段,转化感受态大肠杆菌,挑克隆测序,检测扩增的PD-1启动子片段甲基化状态.结果 与正常对照组(10.8％±4.4％)比较,CHB患者PBMCs上PD-1表达水平明显升高.在CHB患者中,PBMCs上PD-1表达在HBeAg阳性患者(27.1％±18.4％)中显著高于HBeAg阴性患者(19.6％±15.6％),PD-1表达水平与HBV-DNA和ALT水平无明显相关性.PD-1启动子上多个CpG点(-601、-553、-538、-483、-463、-317 bp)甲基化程度与PD-1在单个核细胞上的表达呈负相关.结论 PD-1基因启动子区域甲基化水平可能影响CHB患者PBMCs上PD-1表达.%Objective To observe the expression variations and influencing factors of programmed death one (PD-1) and DNA demethylation of PD-1 promoter on peripheral blood mononuclear cells (PBMCs) in chronic hepatitis B (CHB) patients and further investigate the relationship between the demethylation pattern of PD-1 gene in promoter region and the PD-1 expression on PBMC in CHB patients.Methods A total of 162 subjects,including 144 CHB patients and 18 healthy blood donors,were enrolled.The expression of PD-1 on PBMCs was detected by flow cytometry.And the serum HBV markers,HBV DNA load and liver function were also measured.DNA of PBMCs was treated with sodium bisulfite; the PD-1 promoter fragments were amplified by polymerase chain reaction (PCR) and then transformed into Escherichia coli.Positive clones were selected for sequencing and the methylation status of fragments of PD-1 promoter was examined.Results With the PD-1 expression in normal controls (10.8％ ± 4.4％) as a baseline level,the expression of PD-1 in CHB patients significantly increased.In CHB patients,the serum expression of PD-lin PBMCs from patients with positive HBeAg (27.1％ ± 18.4％) was much higher than that from those with negative HBeAg (19.6％ ± 15.6％).And the expression level of PD-1 was not correlated with serum HBV DNA load and serum level of alanine aminotransferase.The results of bisulfite genomic sequencing showed that demethylation probability of some CG points in PD-1 promoter region (-601,-553,-538,-483,-463,-317 bp) were significantly correlated with PD-1 expression level (P ＜ 0.05).Conclusion The demethylation pattern of PD-1 gene in promoter region is associated with the PD-1 expression on PBMC in CHB patients.