CAJ | 학술논문

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.
목적 탐토만성을형간염(CHB)환자외주혈단개핵세포(PBMCs)상정서성사망수체-1(PD-1)표체화PD-1계동자구역갑기화수평적차이급기영향인소,이급량자지간적관계.방법 사선2009년9월지2011년9월재중남대학상아이의원전염과문진취진144례CHB환자화18명건강헌혈원,류식세포술(FCM)검측PBMCs표면표체PD-1적세포비례,동시진행간공능검측；시간분변형광면역분석법검측CHB환자혈청HBV표지물수평,형광정량PCR검측혈청HBV DNA재량；아류산경납처리PBMCs세포DNA,PCR확증PD-1계동자기인편단,전화감수태대장간균,도극륭측서,검측확증적PD-1계동자편단갑기화상태.결과 여정상대조조(10.8％±4.4％)비교,CHB환자PBMCs상PD-1표체수평명현승고.재CHB환자중,PBMCs상PD-1표체재HBeAg양성환자(27.1％±18.4％)중현저고우HBeAg음성환자(19.6％±15.6％),PD-1표체수평여HBV-DNA화ALT수평무명현상관성.PD-1계동자상다개CpG점(-601、-553、-538、-483、-463、-317 bp)갑기화정도여PD-1재단개핵세포상적표체정부상관.결론 PD-1기인계동자구역갑기화수평가능영향CHB환자PBMCs상PD-1표체.
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.