目的研究纳米二氧化硅(nm—SiO2)对人皮肤表皮细胞系(HaCaT)细胞中聚腺苷二磷酸核糖聚合酶-1(poly[ADP-ribose]polymerase 1,PARP-1)基因的表达及其基因启动子区甲基化的影响。方法分别以2.5、5.0、10.0μg/ml的nm-SiO2溶液和10μg/ml的微米级SiO2(micro-SiO2)溶液处理HaCaT细胞24h;以3μmol/LDNA甲基化转移酶抑制剂5-氮杂-2-脱氧胞苷(5-aza—CdR,DAC)处理48h10μg/ml的HaCaT细胞为阳性对照(DAC组),并设立溶剂对照组(CTRL组)。应用荧光定量聚合酶链式反应(polymerase chain reaction,PCR)和蛋白质印迹法(Western-blot法)检测PARP-1在mRNA水平和蛋白水平的表达变化,应用亚硫酸氢盐测序(bisulfite pyrosequence,BSP)法检测PARP-1基因启动子区CpG岛的甲基化状态。结果HaCaT细胞暴露nm-SiO224h后,经nm-SiO2处理后,micro-SiO2染毒组和2.5μg/ml染毒组PARP-1表达与CTRL组的差异无统计学意义(P〉0.05),与CTRL组比较,5、10μg/ml染毒组及DAC处理组细胞的PARP-1表达下调,差异有统计学意义(P〈0.05),且其下调水平随着浓度的增大而增高。BSP结果显示PARP-1启动子区-229点甲基化状态升高。结论nm-SiO2可以降低HaCaT细胞中PARP-1基因的表达,可能与其对PARP-1基因启动子区CpG岛的甲基化影响有关。
Objective To study the effect of silicon dioxide nanoparticles on the expression and promoter region CpG islands methylation of (Poly [ADP-ribose] polymerase 1, PARP-1 ) gene in human HaCaT Cell. Methods HaCaT Cells were treated with nm-SiO2 at 0, 2.5, 5 and 10 μg/mL and micro-SiO2 at 10 μg/ml for 24 h and DAC treatment was given at 10 μg/ml group for 48 h. Real-time PCR and western blot assay was used to detect the expression of PARP-1 mRNA and protein. BSP (Bisulfite Pyrosequence, BSP)assay was used to detect the promoter region CpG islands methylation status of PARP-1 gene. Results After exposure to nano- SiO2 particles, compared to CTRL group, the mRNA and protein expression of PARP-1 in micro-SlOE and 2.5 μg/ml group unchanged, but he mRNA and protein expression of PARP-1 in 5,10 μg/ml as well as DAC group was down-regulated and there are statistical significance between CTRL group and 5,10 μg/ml as well as DAC group and the PARP-1 promoter region CpG islands showed methylation. Conclusion nano-SiO2 can downregulate PARP-1 expression in HaCaT Cell and this is associated with the change in the methylafion of PARP-1 gene promoter region CpG islands induced by nano-SiO2 particles.