Objective To observe the demethylation effect of demethylation inhibitor 5-aza-cytidine (5-Zac) on programmed death receptor 1 (PD-1) in Molt-4 cells (T lymphocyte cell line) and to investigate the relationship between DNA demethylation and expression of PD-1. Methods Molt-4 cells were cultured in the medium containing different concentrations of 5-Zac(0, 5, 10 μmol/L) for 72 h. According to the concentrations of 5-Zac, the Molt-4 cells were divided into a 0 μmol/L 5-Zac group, a 5 μmol/L 5-Zac group, and a 10 μmol/L 5-Zac group. The expression of PD-1 in Molt-4 cells was detected by flow cytometry and the apoptosis rate was calculated. The mR-NA transcription level of PD-1 was detected by real-time polymerase chain reaction; Molt-4 cell DNA in all groups were treated by sodium bisulfite. The PD-1 promoter fragment was amplified by PCR, the amplification fragments were transformed into E. coli. , the positive clones were selected for equencing, and the methylation status of the fragments of PD-1 promoter was examined. Results Seventy-two hours after the 5-Zac treatment, the expression rate of PD-1 in the Molt-4 cells in the 0 μmol/L 5-Zac group, the 5 μmol/L 5-Zac group, and the 10 μmol/L 5-Zac group was (1.13 ± 0.01 ) % , (18.96 ± 1. 87)% , and (63.09 ±6. 25) % respectively, in a low concentration-dependent way. The PD-1 mRNA expression level was increased significantly with the 5-Zac treatment. Cells apoptosis showed that;compared with the 0 fjimol/L 5-Zac group, the apoptosid rate in the 5 μmol/L 5-Zac group and 10 μmol/L 5-Zac group was signficantly increased, which was ( 1.9 ± 0.06)%, (8.89±1.36)%, and (24.50 ±3.68)% in the 0 μmol/L 5-Zac group, the 5 μmol/L 5-Zac group, and the 10 |xmol/L 5-Zac mol/L group respectively. The bisulfite genomic sequencing showed that the demethylation probability of CpG points on -601 bp and -553 bp was significantly increased in the 5-Zac treated cells compared with those untreated. Conclusion 5-Zac can result in the increase of PD-1 expression in the human lymphoid cell series Molt- 4 in vitro, and the apoptosis rate increases, which is related to PD-1 gene promoter demethylation.%目的:以T淋巴细胞株Molt-4细胞为模型,探讨甲基化抑制剂5-杂氮胞苷(5-azacytidine,5-Zac)对淋巴细胞表面程序性死亡受体-1(programmed death receptor 1,PD-1)基因启动子的去甲基化作用及其诱导的PD-1基因表达的改变,并进一步研究去甲基化作用与PD-1基因表达之间的关系.方法:以不同浓度的5-Zac分组(0 μmol/L组、5μmol/L组、10 μmol/L组)作用于体外培养的Molt-4细胞72 h,流式细胞仪(flow cytometry,FCM)检测细胞表面表达PD-1的Molt-4细胞比例和细胞凋亡率；反转录-聚合酶链反应(reverse transcription polymerase chain reaction,RT-PCR)检测5-Zac作用后PD-1基因mRNA的转录水平；亚硫酸氢钠处理各组Molt-4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:0μmol/L组、5μmol/L组、10 μmol/L组的5-Zac作用于Molt-4细胞72 h后,PD-1在细胞表面的表达率分别为(1.13±0.01)％,(18.96±1.87)％和(63.09±6.25)％,并呈现浓度依赖性；PD-1基因mRNA表达量显著增加；细胞凋亡检测结果显示:与0μmol/L组相比,5μmol/L组、10 μmol/L组5 -Zac处理72 h后Molt-4细胞的凋亡率显著增加,0μmol/L组、5μmol/L组、10μmol/L组凋亡率分别为(1.9±0.06)％,(8.98±1.36)％和(24.5±3.68)％,差异有统计学意义(P＜0.01)；上述3组DNA亚硫酸氢钠测序结果表明:加入甲基化抑制剂5 -Zac处理后,PD-1启动子上-601 bp和-553 bp CpG点去甲基化程度明显增高.结论:甲基化抑制剂5 -Zac可导致体外培养的T淋巴细胞系Molt-4细胞表面PD-1 mRNA表达显著增加,细胞凋亡率增高,这种增高可能与PD-1基因启动子区域出现的去甲基化有关.
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