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目的了解不同剂量的亚砷酸钠(NaAsO2)对人皮肤角质形成细胞(HaCaT细胞)中DNA甲基转移酶1(DNMT1)、组蛋白去乙酰化酶1(HDAC1)基因mRNA转录和蛋白表达的影响。方法分别以0、3.13、6.25、12.5和25μmol/L NaAsO2溶液重复间隔处理HaCaT细胞72 h(NaAsO2处理24 h,隔天再次进行相同处理,共处理3次),以实时荧光定量PCR(Q-PCR)技术检测DNMT1及HDAC1基因的mRNA转录,以免疫印迹分析(western blotting)检测DNMT1及HDAC1蛋白表达。以人表皮鳞癌细胞株A431作为阳性对照。结果细胞经0、3.13、6.25、12.5和25μmol/L NaAsO2溶液处理后,DNMT1 mRNA转录相对表达量分别为0.650 90±0.174 05,0.930 53±0.145 56,0.752 93±0.244 62,0.558 11±0.051 02和0.370 17±0.088 4,差异有统计学意义(F=6.539,P<0.05);HDAC1 mRNA转录相对表达量分别为2.021 80±0.179 57,1.496 98±0.107 55,1.303 24±0.152 08,1.037 75±0.204 88和0.816 74±0.153 12,差异有统计学意义(F=17.914,P<0.05)。DNMT1蛋白相对表达量分别为0.000 87±0.000 10,0.026 04±0.002 63,0.283 19±0.072 62,0.842 61±0.082 39和1.579 87±0.200 83,差异有统计学意义(F=27.799,P<0.05);HDAC1蛋白相对表达量分别为0.034 65±0.012 03,0.273 65±0.012 02,0.634 90±0.239 76,0.775 03±0.126 51和1.126 16±0.167 52,差异有统计学意义(F=9.820,P<0.05)。结论 DNMT1和HDAC1蛋白的高表达是砷致皮肤损害的重要分子事件,这为进一步应用其抑制剂探索砷中毒的防治提供了可能。
Objective To investigate the effects of different doses of sodium arsenite (NaAsO2) on mRNA and protein expression of DNA methyltransferase 1 (DNMT1) and histone deacetylase 1 (HDAC1) genes in human keratinocytes (HaCaT cells) influences. Methods HaCaT cells were treated with 0, 3.13, 6.25, 12.5 and 25μmol / L NaAsO2 solution for 72 h (NaAsO2 treatment for 24 h, the same treatment was carried out again on the next day for 3 times), and real-time fluorescent quantitative PCR PCR) was used to detect the mRNA transcripts of DNMT1 and HDAC1. The expression of DNMT1 and HDAC1 protein was detected by western blotting. Human epidermal squamous cell carcinoma cell line A431 was used as a positive control. Results After treated with 0, 3.13, 6.25, 12.5 and 25μmol / L NaAsO2 solution, the relative expression of DNMT1 mRNA was 0.650 90 ± 0.174 05,0.930 53 ± 0.145 56,0.752 93 ± 0.244 62,0.558 11 ± 0.051 02 And 0.370 17 ± 0.088 4 respectively (F = 6.539, P <0.05). The relative expression levels of HDAC1 mRNA were 2.021 80 ± 0.179 57,1.496 98 ± 0.107 55,1.303 24 ± 0.152 08,1.037 75 ± 0.204 88 and 0.816 74 ± 0.153 12, the difference was statistically significant (F = 17.914, P <0.05). The relative expression levels of DNMT1 protein were 0.000 87 ± 0.000 10,0.026 04 ± 0.002 63,0.283 19 ± 0.072 62,0.842 61 ± 0.082 39 and 1.579 87 ± 0.200 83, respectively, the difference was statistically significant (F = 27.799, P <0.05 ); The relative expression of HDAC1 protein was 0.034 65 ± 0.012 03,0.273 65 ± 0.012 02,0.634 90 ± 0.239 76,0.775 03 ± 0.126 51 and 1.126 16 ± 0.167 52, respectively, the difference was statistically significant (F = 9.820, P <0.05). Conclusion The high expression of DNMT1 and HDAC1 proteins is an important molecular event of arsenic-induced skin lesions, which may provide further evidence for the prevention and treatment of arsenic poisoning by using their inhibitors.