砷诱导L-02细胞的耐砷性及细胞内<i>MDR1、GST-π</i>基因的表达

申旭波; 周远忠; 姜慧; 贾菲菲; 邹焰

砷诱导L-02细胞的耐砷性及细胞内MDR1、GST-π基因的表达

Expression of MDR1,GST-π and Arsenic Tolerance of L-02 Cell after 6 Weeks Culture with Low Concentration of Arsenic

摘要

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、100 mmol/L的亚砷酸钠(NaAsO₂)作用 24 h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-time PCR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 10 mmol/L的 NaAsO₂作用 24 h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。

Abstract:
Objective To study the expression of multidrug resistance 1(MDR1)and glutathione S-transferase-π(GST-π) in acquired arsenic tolerant L-02 hepatic cells, and to discuss its relationship with the acquired tolerance 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 24 h 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 contro(l normal cells). Induced cells and normal cells were cultured under the same condition for 6 weeks, then the survival rate and LC₅₀ of induced cells and normal cells were detected by MTT 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-glycoprotein(P-gp), GST-π was examined by immunohistochemical SABC. Cell survival rate and intracellular concentrations of total arsenic of the cells that were cultured 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 < .001); expression of the genes for GST-π/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 < 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 hepatocyte cell line. This may provide an experimental basis for the prevention and treatment of arsenic poisoning at the gene level.

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