CAJ | 학술논문

目的：研究5-氮杂胞苷逆转P16基因甲基化对血管瘤细胞增殖和凋亡的影响。方法采用BSP法检测未处理组和5-氮杂胞苷处理组EOMA小鼠血管瘤细胞P16基因启动子甲基化情况，RT-PCR和Western Blot分别检测P16基因mRNA和蛋白质表达，采用流式细胞仪检测细胞增殖、细胞周期和细胞凋亡，比较2组P16基因启动子甲基化、mRNA和蛋白质表达、细胞增殖、细胞周期和凋亡的差异。结果5-氮杂胞苷处理组EOMA小鼠血管瘤细胞P16基因启动子第1～13CG位点甲基化率均为0％，明显低于未处理组细胞；处理组P16基因mRNA和蛋白质表达均显著高于未处理组细胞（P＜0.05）；处理组细胞吸光度、S期、G2/M期和PI显著低于未处理细胞（P＜0.05），G0/G1期和凋亡率显著高于未处理组细胞（P＜0.05）。结论血管瘤细胞P16基因处于高甲基化和表达沉默状态。5-氮杂胞苷可逆转血管瘤细胞P16基因启动子甲基化状态，使沉默的P16基因重新获得表达而抑制血管瘤细胞增殖并促进其凋亡。
목적：연구5-담잡포감역전P16기인갑기화대혈관류세포증식화조망적영향。방법채용BSP법검측미처리조화5-담잡포감처리조EOMA소서혈관류세포P16기인계동자갑기화정황，RT-PCR화Western Blot분별검측P16기인mRNA화단백질표체，채용류식세포의검측세포증식、세포주기화세포조망，비교2조P16기인계동자갑기화、mRNA화단백질표체、세포증식、세포주기화조망적차이。결과5-담잡포감처리조EOMA소서혈관류세포P16기인계동자제1～13CG위점갑기화솔균위0％，명현저우미처리조세포；처리조P16기인mRNA화단백질표체균현저고우미처리조세포（P＜0.05）；처리조세포흡광도、S기、G2/M기화PI현저저우미처리세포（P＜0.05），G0/G1기화조망솔현저고우미처리조세포（P＜0.05）。결론혈관류세포P16기인처우고갑기화화표체침묵상태。5-담잡포감가역전혈관류세포P16기인계동자갑기화상태，사침묵적P16기인중신획득표체이억제혈관류세포증식병촉진기조망。
Objective To study the effects of 5-azacytidine’s demethylation for P16 gene on hemangioma cell’s proliferation and apoptosis.Methods Bisulfite sequencing PCR was applied to detect P16 gene′s promoter methylation status in 5-azacytidine treated and untreated EOMA cell line.RT-PCR and western blot were used to detect the P16 gene mRNA and protein expressions.Flow cytometry was used to detect cell proliferation, cell cycle and cell apoptosis.The differences of P16 gene′s promoter methylation status,mRNA and protein expressions,cell proliferation,cell cycle and apoptosis in two groups were compared. Results The methylation rates in 1st and 13th CGs were 0%after 5-azacytidine treatment in EOMA hemangioma cell line,which were lower than in untreated cells.The mRNA and protein expressions increased after 5-azacytidine treatment,which were significantly higher than in untreated cells.The absorbance,S phase and G2/M phase and PI after 5-azacytidine treatment were lower than untreated cells,while the G0/G1 phase and apoptosis rates were higher.Conclusion The P16 gene promoter is hypermethylated in hemangioma cells with silent gene expressions.5-azacytidine could reverse P16 gene′s promoter methylation and silent gene expressions,which inhibit hemangioma cell’s proliferation and promote apoptosis.