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目的探讨多药耐药相关蛋白1(MRP1)与人肝细胞(L-02)耐砷性之间的关系。方法采用人肝细胞L-02细胞株,噻唑兰法(MTT)进行24 h急性NaAsO2毒性试验,选择细胞生存率在90%～95%时的NaAsO2浓度为诱导浓度,同时设1组不加砷诱导的L-02细胞作为同步对照。置于细胞培养箱中37℃,5%CO2常规培养6周,定期换液传代,每周用MTT法计算半数致死浓度(LC50)及细胞的生存率;6周后,将加砷诱导的L-02肝细胞与同步对照细胞在培养基中培养至细胞80%融合,然后进行24 h急性砷毒性试验(NaAsO2终浓度为0、2.5、5.0、10μmol/L);采用real-time PCR检测细胞内MRP1mRNA的表达情况;MTT法检测细胞的存活率;石墨炉原子吸收光谱法检测各组细胞内总砷浓度。结果实验组细胞MRP1mRNA的表达明显高于对照组(P<0.05);24 h急性砷毒性试验中,实验组不同砷浓度下细胞生存率和LC50明显高于对照组(均P<0.001);实验组细胞内总砷浓度明显较同步对照组低(P<0.001)。结论L-02肝细胞具有可诱导的对砷的耐受性,其耐砷性与MRP1高表达有关。
Objective To investigate the relationship between multidrug resistance-associated protein 1 (MRP1) and arsenic tolerance of human hepatocytes (L-02). Methods 24 h acute NaAsO2 toxicity test was performed on L-02 cell line of human hepatocytes and MTT. The concentration of NaAsO2 was chosen as the induced concentration when the cell survival rate was 90% ~ 95%. Meanwhile, Induced L-02 cells served as synchronous controls. Placed in a cell incubator at 37 ℃, 5% CO2 routine culture for 6 weeks, regular liquid change passage, weekly MTT method to calculate the median lethal concentration (LC50) and cell survival rate; 6 weeks after adding arsenic-induced L -02 hepatocytes and synchronous control cells were cultured in culture medium to 80% confluency, then subjected to 24 h acute arsenic toxicity test (final concentration of NaAsO2 0,2.5,5.0,10μmol / L); cells were detected by real-time PCR The expression of MRP1mRNA, the survival rate of cells was detected by MTT method, and the total arsenic concentration in each group was detected by graphite furnace atomic absorption spectrometry. Results The expression of MRP1 mRNA in the experimental group was significantly higher than that in the control group (P <0.05). In the 24 h acute arsenite toxicity test, the cell viability and the LC50 of the experimental group were significantly higher than those of the control group (all P <0.001) The total intracellular total arsenite concentration was significantly lower than the synchronous control group (P <0.001). Conclusion L-02 hepatocytes have an inducible resistance to arsenic, which is related to the high expression of MRP1.