CAJ | 학술논문

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-Zac)对淋巴细胞表面程序性死亡受体-1(PD-1)基因肩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(0、5、10μmoL/L)作用于体外培养的Molt-4细胞72 h,流式细胞术(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(RT-PCR)检测5-Zac作用后PD-1基因mRNA的转录水平;亚硫酸氧钠处理各组Molt-4细胞DNA,PCR扩增与转录因子Brn-2结合的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 0、5、10μmol/L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%4±0.01%、18.96%±1.87%、63.09%±6.25%,并呈现浓度依赖性(P<0.05);PD-1基因mRNA表达量显著增加;细胞凋亡检测结果显示与未处理组相比,5-Zac处理72 h后Molt-4细胞的凋亡率显著增加,3组凋亡率分别为1.9%4±0.06%、8.98%4±1.36%、24.5%4±3.68%,差异有统计学意义(P<0.01);3组DNA亚硫酸氢钠测序结果表明,5-Zac处理后,与转录因子Bin-2结合位置的CG点明显受到去基化影响,其去甲基化概率与周围其他CG点去甲基化概率比较差异有统计学意义(P<0.05).结论甲基化抑制剂5-Zac可导致体外培养的淋巴细胞系Molt-4细胞表面PD-1表达显著增加,PD-1基因mRNA表达增加,细胞凋亡率增高;PD-1基因活化和表达的增加可能与5-Zac降低PD-1基因启动子上与转录因子Bin-2结合位置的CG点甲基化水平,从而促进Brn-2与基因启动子结合,使转录表达增加有关.
목적 이T림파세포주Molt-4세포위모형,탐토갑기화억제제5-잡담포감(5-Zac)대림파세포표면정서성사망수체-1(PD-1)기인견동자적거갑기화작용급기유도적PD-1기인표체적개변,병진일보연구거갑기화작용여PD-1기인표체지간적관계.방법 이불동농도적5-Zac(0、5、10μmoL/L)작용우체외배양적Molt-4세포72 h,류식세포술(FCM)검측세포표면표체PD-1적Molt-4세포비례화세포조망솔;반전록-취합매련반응(RT-PCR)검측5-Zac작용후PD-1기인mRNA적전록수평;아류산양납처리각조Molt-4세포DNA,PCR확증여전록인자Brn-2결합적PD-1계동자기인편단,전화감수태대장간균,도극륭측서,검측확증적PD-1계동자편단갑기화상태.결과 0、5、10μmol/L적5-Zac작용우Molt-4세포72 h후,PD-1재세포표면적표체솔분별위1.13%4±0.01%、18.96%±1.87%、63.09%±6.25%,병정현농도의뢰성(P<0.05);PD-1기인mRNA표체량현저증가;세포조망검측결과현시여미처리조상비,5-Zac처리72 h후Molt-4세포적조망솔현저증가,3조조망솔분별위1.9%4±0.06%、8.98%4±1.36%、24.5%4±3.68%,차이유통계학의의(P<0.01);3조DNA아류산경납측서결과표명,5-Zac처리후,여전록인자Bin-2결합위치적CG점명현수도거기화영향,기거갑기화개솔여주위기타CG점거갑기화개솔비교차이유통계학의의(P<0.05).결론 갑기화억제제5-Zac가도치체외배양적림파세포계Molt-4세포표면PD-1표체현저증가,PD-1기인mRNA표체증가,세포조망솔증고;PD-1기인활화화표체적증가가능여5-Zac강저PD-1기인계동자상여전록인자Bin-2결합위치적CG점갑기화수평,종이촉진Brn-2여기인계동자결합,사전록표체증가유관.
Objective To investigate the demethylation and changes in gene expression of programmed death receptor-1 ( PD-1) caused by methylation inhibitor 5- azacytidine (5-Zac) in lymphocyte series Molt-4 cells and its mechanism. Methods Molt-4 cells were cultured in different concentrations of 5-Zac(0, 5, 10 Umol/L)for 72 h, ratio of cell expressing PD-1 and apoptosis rate were detected by FCM, transcription of PD-1 gene mRNA was detected by RT-PCR. Molt-4 cell DNA of all groups were disposed by sodium bisulfite, PD-1 gene promoter fragment binded with transcription factor Brn-2 was amplified by PCR,these amplification fragments were transformed into E. coli. Positive clones were selected by sequencing,methylation status of the fragments binded with transcription factor Brn-2 was examined. Results S-Zac could increase the PD-1 expression of Molt-4 cells. PD-1 expression rate in 0 μmol/L 5-Zac( 1. 13%±0.01% ) treated cells was found more lower than that in both 5 μmol/L and 10 μmol/L 5-Zac treated cells (18. 96% ±1. 87% , 63. 09% ± 6. 25% , P < 0. 05 ) , and they showed concentration-dependent (P <0.01). Cells apoptosis rate and PD-1 mRNA expression were also observed increased significantly with 5-Zac treating. Demethylation probability of CG points showed significant difference between transcription factor Brn-2 binding site and other four locations (P < 0.05 ). Conclusion 5 -Zac inhibits cell grouth in human lymphoid cell series Molt-4 by inducing PD-1 gene expression and promoter demethylation. PD-1 gene promoter binding transcription factor Brn-2 fragment CG point demethylation may be one of the important mechanisms in 5-Zac treated Molt-4 cells.