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目的观察亚砷酸钠染毒对人肝细胞(L02)凋亡及其机制的探讨。方法采用人肝细胞L02细胞株,亚砷酸钠染毒剂量范围为0~40μmol/L,处理时间为24和48 h。MTT法检测细胞增殖,并将活力为80%以上作为染毒剂量,选择亚砷酸钠终浓度为0、1.0、5.0和10μmol/L,处理24h。Hochest33258染色法观察细胞凋亡,CM-H2DCFDA荧光染色法观察胞内ROS形成,Western bloting检测NF-κB信号通路p-p65及p65蛋白的表达,用NAC干预2 h,加亚砷酸钠5.0μmol/L处理后,进一步观察上述指标。结果随着砷染毒剂量的增加,细胞增殖呈现先增高后降低的趋势,凋亡细胞数和胞内ROS则呈现逐步增多的现象,但NAC预处理后ROS产生及凋亡细胞数目则显著降低,提示氧化应激在砷致人肝细胞L02中发挥作用;p-p65蛋白表达水平随砷染毒剂量的增加而增加,NAC预处理可降低其形成,以上差异均有统计学意义。结论亚砷酸钠可经由氧化应激引起人肝细胞L02发生凋亡,且激活NF-κB信号通路有关。
Objective To investigate the apoptosis of human hepatocytes (L02) induced by sodium arsenite and its mechanism. Methods The human hepatocyte L02 cell line was used. The dose of sodium arsenite was 0-40 μmol / L and the treatment time was 24 and 48 h. Cell proliferation was detected by MTT assay. The viability was above 80%. The final concentration of sodium arsenite was 0, 1.0, 5.0 and 10 μmol / L for 24 h. Hochest33258 staining was used to observe the cell apoptosis. The intracellular ROS was detected by CM-H2DCFDA staining. The expressions of p-p65 and p65 were detected by Western blotting. The cells were treated with NAC for 5.0 h / L treatment, further observation of the above indicators. Results With the increase of arsenic dose, the cell proliferation increased at first and then decreased. The number of apoptotic cells and intracellular ROS showed a gradual increase. However, the production of ROS and the number of apoptotic cells were significantly decreased after NAC pretreatment , Suggesting that oxidative stress played a role in arsenic-induced human hepatocyte L02. The expression level of p-p65 protein increased with the increase of arsenic dose, and NAC pretreatment could reduce the formation of these cells. The above differences were statistically significant. Conclusion Sodium arsenite can induce L02 apoptosis in human hepatocytes via oxidative stress, and activate NF-κB signaling pathway.