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目的研究二溴乙腈(dibromoacetonitrile,DBAN)对3种人源性肝细胞的氧化应激作用。方法取处于对数生长期的人肝癌(Hep G2)细胞、人正常肝(Chang Liver)细胞和人胚胎肝(L-02)细胞,分别暴露于含终浓度为0(对照)、0.5、1、5、10、20、50、100μmol/L DBAN染毒溶液中孵育24 h。检测细胞活性及细胞内活性氧(ROS)、总谷胱甘肽(GSH)含量和超氧化物歧化酶(SOD)活力。将稳定转染ARE荧光素酶报告基因质粒的Hep G2细胞分别暴露于0(对照)、0.5、1、5、10μmol/L DBAN染毒溶液孵育6 h,测定抗氧化响应元件(ARE)报告基因活性。结果与对照组比较,50、100μmol/L DBAN暴露组Hep G2细胞及100μmol/L DBAN暴露组Chang Liver细胞和L-02细胞的存活率均较低,而1、5μmol/L DBAN暴露组Chang Liver细胞的存活率均较高,差异均有统计学意义(P<0.05);且随着DBAN暴露浓度的升高,3种细胞的存活率均呈先上升后下降的趋势。与对照组比较,50、100μmol/L DBAN暴露组Hep G2细胞和L-02细胞及各浓度DBAN暴露组Chang Liver细胞内ROS的含量均较高,差异均有统计学意义(P<0.05);随着DBAN暴露浓度的升高,3种细胞内ROS的含量均呈上升趋势。与对照组比较,10、20μmol/L DBAN暴露组Hep G2细胞和10μmol/L DBAN暴露组Chang Liver细胞及各浓度DBAN暴露组L-02细胞内GSH的含量均较高,差异均有统计学意义(P<0.05);随着DBAN暴露浓度的升高,3种细胞内GSH的含量呈先上升后下降趋势。与对照组比较,10、20μmol/L DBAN暴露组Hep G2细胞及20μmol/L DBAN暴露组Chang Liver细胞内SOD活性均较低,差异均有统计学意义(P<0.05);而各浓度DBAN暴露组L-02细胞内的SOD活力均无明显改变;随着DBAN暴露浓度的升高,3种细胞内SOD的活性总体呈先上升后下降趋势。与对照组比较,5、10μmol/L DBAN暴露组Hep G2-ARE报告基因活性均较高,差异有统计学意义(P<0.05);随着DBAN暴露浓度的升高,Hep G2-ARE报告基因的活性均呈上升趋势。结论 DBAN可通过氧化应激对3种人源性肝细胞产生较强的细胞毒性;其中,Chang Liver细胞对DBAN毒性更为敏感。
Objective To investigate the oxidative stress of three human hepatocytes induced by dibromoacetonitrile (DBAN). Methods Hep G2 cells, human normal liver cells and human embryonic liver (L-02) cells in logarithmic growth phase were harvested and exposed to medium with final concentrations of 0 (control), 0.5, 1 , 5,10,20,50,100μmol / L DBAN exposure solution for 24 h. Cell viability, intracellular reactive oxygen species (ROS), total glutathione (GSH) and superoxide dismutase (SOD) activity were measured. Hep G2 cells stably transfected with ARE luciferase reporter plasmids were exposed to 0 (control), 0.5, 1, 5, and 10 μmol / L DBAN for 6 h respectively, and the antioxidant response element (ARE) reporter gene active. Results Compared with the control group, the survival rates of Chang Liver cells and L-02 cells in 50,100μmol / L DBAN exposure group and 100μmol / L DBAN exposure group were lower than those in DBAN exposure group, while those in 1,5 μmol / L DBAN exposure group (P <0.05). With the increase of DBAN exposure, the survival rates of all three kinds of cells increased first and then decreased. Compared with the control group, the content of ROS in the Hep G2 and L-02 cells exposed to 50,100μmol / L DBAN exposure group and the Chang Liver cells exposed to various concentrations of DBAN were higher than those in the control group (P <0.05). With the increase of DBAN exposure, the content of ROS in all three kinds of cells showed an upward trend. Compared with the control group, the content of GSH in 10,2 μmol / L DBAN exposure group and Chang Liver cells in 10 μmol / L DBAN exposure group and L-02 cells in each concentration of DBAN exposure group were higher, the differences were statistically significant (P <0.05). With the increase of DBAN exposure concentration, the content of GSH in three kinds of cells first increased and then decreased. Compared with the control group, the SOD activity in Hep G2 cells exposed to 10,20 μmol / L DBAN exposure group and Chang Liver cells exposed to 20 μmol / L DBAN was lower than that of the control group, the difference was statistically significant (P <0.05); while DBAN exposure There was no significant change in SOD activity in L-02 cells. With the increase of DBAN exposure, the activity of SOD in all three kinds of cells increased firstly and then decreased. Compared with the control group, the reporter gene activity of Hep G2-ARE in 5 and 10μmol / L DBAN exposure groups was significantly higher (P <0.05). With the increase of DBAN exposure, the Hep G2-ARE reporter gene The activities of all showed an upward trend. Conclusions DBAN can induce cytotoxicity on three kinds of human hepatocytes through oxidative stress. Among them, Chang Liver cells are more sensitive to DBAN toxicity.