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

Demethylation pattern of PD-1 gene in promoter region is associated with the PD-1 expression onperipheral blood mononuclear cells in chronic hepatitis B patients

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TL=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HBVDNA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AIJT水平无明显相关性。PD-1启动子上多个CpG点（-601、-553、-538、-483、-463、-317bp）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-I 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 coll. 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 bisulfitegenomic 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.