谷胱甘肽S转移酶-π与人肝细胞耐砷性关系的研究

Correlation of GST-π and Arsenic tolerance in human hepatocyte cell

目的探讨谷胱甘肽S转移酶-π(GST-π)在人胚肝细胞(L-02细胞)耐砷性中的作用。方法培养L-02细胞和耐砷L-02细胞,用实时PCR和免疫组织化学法检测细胞GST-πmRNA和蛋白表达情况;两种细胞均采用亚砷酸钠(NaAsO2)2.5、5.0、10 mmol/L及NaAsO2与GST-π抑制剂依他尼酸(EA)联合培养24 h,噻唑蓝(MTT)法和石墨炉原子吸收光谱法检测细胞生存率和细胞内砷浓度。结果耐砷L-02细胞GST-πmRNA及蛋白表达阳性率均显著高于L-02细胞(P<0.001);单用NaAsO2培养时,耐砷L-02细胞生存率明显高于L-02细胞、砷浓度明显低于L-02细胞(P<0.001);NaAsO2与EA联用时两种细胞内砷浓度增加、细胞存活率降低(P<0.001)。结论L-02细胞的耐砷性可能与GST-π基因高表达有关,此从基因水平上为砷中毒的防治提供了实验依据。

Objective To investigate the role of glutathione S transferase -π （GST-π） in the Arsenic tolerance of human embryo hepatocyte cell （L-02 cell）. Methods L-02 cells and Arsenic-resistant L-02 cells were cultured, then the levels of GST-π mRNA was determined by real-time quantitative -PCR,and the expression of GST-π protein was examined by immunohistochemical method. Then the above cells were incubated with fresh medium containing 2.5,5. 0, 10 μmol/L NaAsO2 in the presence or absence of EA（ ethacrynie acid, an inhibiteor for GST-π） for 24 hs, and the cell survival rate and total arsenic was determined by using 3-[4,5-dimethy Ithiazol-2-yl]-2,5-diphenyl tetrzolium（MTT） and graphite furnaee atomic absorption spectrometry. Results The expression of GST-π mRNA and protein in Arsenic-resistant L-02 cells were higher than those in L-02 cells （P 〈 0.001 ）. When cultured with NaAsO2 alone, the survival rate of Arsenic-resistant L-02 cells was apparently higher than that of L-02 cells, and the cellular Arsenic content was markedly lower than that of L- 02 cells （P 〈 0. 001 ）. When cultured with NaAsO2 and EA,the Arsenie content in both kinds of cells increased, while the survival rate decreased （P 〈 0.001 ）. Conclusions The Arsenic tolerance of L-02 cells may be correlated with the overexpression of GST-π, which provides experimental basis for the prevention and treatment of arsenic poisoning at gene level.