多药耐药相关蛋白1与人肝细胞耐砷性关系的研究

Overexpression of MRP1 Is Associated with Acquired Tolerance To Arsenic In L-02 Cell

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 study the relativity of Multidrug resistance associated protein 1 （ MRPI ） gene expression and Acquired tolerance to arsenic in human hepatocyte cell line （ L - 02 ）. Methods Human hepatoeyte cell line （ L - 02 ） were ex- posed to low - level arsenite for 6 weeks, 3 - [4,5-dimethy Ithiazol - 2 - yl] - 2,5 - diphenyl tetrzolium （ MTT ） was used to de- tect the survival rate and LC50 of arsenic - exposed cells and the control which can reflect the change of arsenic tolerance. After 24 h cytotoxicity test,we chose the initial dose under which the cell survival rate was 90% to 95% to eongtinuously induce for a- bout 6 weeks. Parallel cultures grown in Medium without arsenic provided passage - matched control. 6 weeks later, cells contin- uously exposed to low -level sodium arsenite and cells passage -matched control were geown to 80% congfluence in medium, and then cells were incubated with fresh medium containing 0,2.5,5.0,10 μmol/L NaAsO2. 24 h later, The levels of MRP1 mRNA were determined by real - time quantitative PCR, cellular total arsenic was determined using graphite furnace atomic absorption spectrometry. Results Real - time quantitative PCR analysis confirmed increased expression of the genes for MRP1 in arsenite - exposed cells （ P 〈 0.05 ）. Cells continuously exposed to low level sodium arsenite exhibited dramatic resistance to acute arse- nite toxicity after six weeks, the LC50 and survival rate of arsenite - exposed L - 02 cells was apparently higher （ P 〈 0. 001 ）. cellular arsenic content was markedly decreased in arsenitr - exposed cells （ P 〈 0. 001 ）. Conclusions Human hepatocyte cell line L-02 possess induced to arsenic tolerance phenomenon ,Acquired tolerance to arsenic is associated with increased expression MRP1 in human hepatocyte cell line.