摘要
[目的]观察砷诱导人胚肝(L-02)细胞耐砷性及细胞内耐砷性多药耐药基因1(MDR1)、谷胱甘肽-s-转移酶π(GST-π)的表达水平。[方法]应用噻唑蓝(MTT)比色法检测浓度分别为0、1、10、20、30、40、50、60、70、80、90、100mmol/L的亚砷酸钠(NaAsO2)作用24h后,对L-02细胞生存率的影响。并选择细胞生存率在90%～95%时的NaAsO2浓度为诱导浓度对细胞进行培养,以不加砷诱导的L-02细胞为对照,两组细胞在相同条件下培养6周,利用MTT法每周观察细胞生存率并计算半致死浓度(LC50)来反映细胞对砷的耐受性改变;利用real-timePCR检测培养6周后的细胞内MDR1、GST-π mRNA的表达情况;免疫组化法(SABC法)检测细胞内P-糖蛋白(P-gp)、GST-π蛋白的表达情况。利用MTT法和石墨炉原子吸收光谱法检测浓度为10mmol/L的NaAsO2作用24h后细胞生存率和细胞内总砷浓度以观察两组细胞在再次大剂量急性砷中毒中的反应。[结果]经NaAsO2诱导6周后,诱导细胞LC50和生存率都明显高于正常细胞(P<0.001);诱导细胞MDR1、GST-π mRNA的表达明显较正常细胞高(P<0.001);与对照组比较,诱导细胞P-gp、GST-π蛋白表达明显增高(P<0.001);在再次急性砷中毒试验中,诱导细胞生存率明显高于正常细胞(P<0.001),诱导细胞内总砷浓度较正常细胞低(P<0.001)。[结论]L-02细胞具有可诱导的对砷的耐受现象,其耐砷性可能与MDR1、GST-π基因高表达有关。
[ Objective ] To study the expression of muhidrug resistance 1 （ MDR1 ） and glutathione S-transferase-π （ GST-π ） in acquired arsenic tolerant L-02 hepatic cells, and to diseuss its relationship with the acquired toleranee to arsenic. [ Methods ] MTT （ 3- [ 4, 5-dimethy thiazol-2-yl ] -2, 5-diphenyl tetrazolium ） assay was conducted to detect the effects of NaAsO2 exposure in various concentrations（ 0, 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μmol/L ）for 24h on the survival rate of L-02 cells, then the initial dose under which the cell survival rate was 90%-95% was chosen to be added into the medium for culturing experiment L-02 cells（ induced cells ）. L-02 cells grown in medium without arsenic were provided as eontrol（ normal cells ）. Indueed eells and normal cells were cultured under the same condition for 6 weeks, then the survival rate and LC50 of indueed cells and normal cells were detected by MTr assay which can reflect the change of arsenic tolerance. The levels of MDR1 and GST-π mRNA were determined by real-time quantitative PCR, and the expression of P-glyeoprotein （ P-gp ）, GST-π was examined by immunohistoehemieal SABC. Cell survival rate and intraeellular concentrations of total arsenic of the cells that were euhured with 10 μmol/L NaAsO2 for 24 h were measured in order to observe the response of the cells to another large dose acute arsenic exposure. [ Results ] After 6 weeks after induction of NaAsO2, LC50 and survival rate of induced cells were significantly higher than that of normal cells（ P 〈 0.001 ）; expression of the genes for GST-n/MDR1 in induced cells was significantly higher than that in normal cells（ P 〈 0.001 ）. Compared with normal cells, expression of P-pg/GST-π protein in induced cells was significantly higher（ P 〈 0.001 ）. In the re-trial of acute arsenic poisoning, the survival rate of induced cells was significantly higher than that of normal cells（ P 〈0 .001 ）, the total cellular arsenic content was markedly decreased in induced cells（ P 〈 0.001 ）. [ Conclusion ] L-02 cells possess induced arsenic tolerance phenomenon. Acquired tolerance to arsenic is associated with increased expression of MDR1/GST-π in human hepatoeyte cell line. This may provide an experimental basis for the prevention and treatment of arsenic poisoning at the gene level.
出处
《环境与职业医学》
CAS
北大核心
2010年第3期135-137,141,共4页
Journal of Environmental and Occupational Medicine
基金
贵州省科技厅项目[编号:C-271(2005)2059]
