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目的探讨慢性砷暴露对人皮肤角质形成细胞(human keratinocytes,HaCaT)药物转运蛋白mRNA表达和砷代谢的影响。方法将HaCaT细胞随机分为慢性对照(不予以砷处理)组和慢性砷暴露(100 nmol/L亚砷酸钠染毒28周)组,培养于含10%胎牛血清和1%双抗的培养基中,收集处于对数生长期的细胞,采用实时荧光定量(real-time quantitative PCR)法检测细胞多药耐药相关蛋白mRNA的表达水平。将处于对数生长期的HaCaT对照细胞和慢性砷暴露细胞分别暴露于含终浓度为0(对照)、2.5、5、10、20、30、40、50、60、80、100、150μmol/L亚砷酸钠的培养基中孵育24 h,采用MTS法检测细胞活性。将慢性砷暴露组细胞和对照细胞予以10μmol/L亚砷酸钠处理24 h,采用氢化物发生-原子吸收分光光度法检测细胞砷蓄积和砷排放量。结果与对照细胞相比,慢性砷暴露HaCaT细胞多药耐药相关蛋白ABCC2和ABCC4 mRNA表达水平均升高,差异有统计学意义(P<0.05);而ABCC1、ABCC3、ABCC5、ABCG1、ABCG2 mRNA的表达水平均无显著变化。与对照细胞相比,慢性砷暴露HaCaT经各浓度急性砷处理后细胞存活率均升高,差异有统计学意义(P<0.05)。与对照细胞相比,10μmol/L亚砷酸钠染毒24 h后,慢性砷暴露细胞内的砷蓄积量降低,而砷排放量升高,差异均有统计学意义(P<0.05)。结论慢性砷暴露的HaCaT细胞药物转运蛋白mRNA的表达升高,砷排出能力增强,对砷毒性产生耐受性。
Objective To investigate the effect of chronic arsenic exposure on mRNA expression of human keratinocytes (HaCaT) and arsenic metabolism. Methods HaCaT cells were randomly divided into the chronic control group (without arsenic treatment) and the chronic arsenic exposure group (exposed to 100 nmol / L sodium arsenite for 28 weeks). The cells were cultured in 10% fetal bovine serum and 1% The cells in logarithmic growth phase were collected, and the expression of multidrug resistance-related protein mRNA was detected by real-time quantitative PCR. HaCaT control cells and chronic arsenic exposed cells in logarithmic growth phase were exposed to a final concentration of 0 (control), 2.5, 5, 10, 20, 30, 40, 50, 60, 80, 100, 150 micromol / L Sodium arsenite culture medium for 24 h, using MTS assay cell activity. Chronic arsenic exposed cells and control cells were treated with 10 μmol / L sodium arsenite for 24 h, and the arsenic accumulation and arsenic emissions of cells were measured by hydride generation - atomic absorption spectrophotometry. Results Compared with the control cells, the expression levels of ABCC2 and ABCC4 mRNA in HaCaT cells exposed to chronic arsenic were significantly increased (P <0.05), while the expressions of ABCC1, ABCC3, ABCC5, ABCG1 and ABCG2 mRNA No significant changes in the expression levels. Compared with the control cells, the cell viability of HaCaT exposed to Arsenic in various concentrations increased after chronic arsenic exposure, the difference was statistically significant (P <0.05). Compared with the control cells, arsenic accumulation in 10μmol / L sodium arsenite decreased in chronic arsenic exposed cells and increased in arsenic after 24 hours of exposure. The difference was statistically significant (P <0.05). Conclusion The expression of drug transporter mRNA in HaCaT cells exposed to chronic arsenic is increased, and the arsenic excretion ability is enhanced.