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目的观察Hep G2、Hep3B、Hu H7和SMCC-7721细胞对亚砷酸钠的反应,CD133及CD13分子的表达和肿瘤球形成的差异,分析低浓度亚砷酸钠处理前后Hu H7和原代HCC细胞Oct4、Sox2因子的表达变化,探讨低浓度亚砷酸钠对HCC细胞生长的抑制机制。方法 CCK-8法检测0、0.1、0.2、0.4、0.6、0.8、1.0μg/m L不同浓度亚砷酸钠处理48、72、96 h对Hep G2、Hep3B、Hu H7和SMCC-7721细胞生长的抑制作用;分析Hep G2、Hep3B、Hu H7、SMCC-7721和原代HCC细胞CD13、CD133表达及肿瘤球形成能力,确定是否含肝癌干细胞(LCSCs);观察0.5μg/m L亚砷酸钠处理Hu H7和原代HCC细胞前后Oct4和Sox2基因和蛋白表达的变化,分析低浓度亚砷酸钠对LCSCs有何作用。结果亚砷酸钠处理HCC细胞48、72、96 h,亚砷酸钠明显抑制Hep G2、Hep3B、Hu H7和SMCC-7721细胞生长,对HCC细胞增殖抑制作用与浓度和作用时间有关。亚砷酸钠处理HCC细胞96 h,0.2、0.4μg/m L亚砷酸钠已经明显抑制SMCC-7721细胞生长(P<0.05,P<0.01)。0.4μg/m L亚砷酸钠明显抑制Hep G2(P<0.01)和Hep3B(P<0.01)细胞增殖。0.1、0.2、0.4、0.6μg/m L亚砷酸钠刺激Hu H7细胞增殖,但差异无统计学意义(P>0.05),0.8μg/m L亚砷酸钠非常明显抑制Hu H7细胞增殖(P<0.01)。亚砷酸钠对Hu H7细胞存在低浓度刺激Hu H7细胞增殖和较高浓度抑制Hu H7细胞增殖的双向作用,随着作用时间延长,相同浓度下抑制作用增强。低浓度亚砷酸钠对SMCC-7721、Hep G2和Hep3B细胞几乎无刺激增殖作用。Hep G2、Hep3B和SMCC-7721既不表达CD133,也不形成肿瘤球。Hu H7和原代HCC细胞同时表达CD133、CD13标志,也具有形成肿瘤球能力,Hu H7和原代HCC细胞含有CD133+CD13+LCSCs。Hu H7和原代HCC细胞表达Oct4和Sox2基因和蛋白,0.5μg/m L亚砷酸钠可以下调Hu H7和原代HCC细胞Oct4和Sox2基因和蛋白表达。结论低浓度亚砷酸钠下调或抑制含CD133+CD13+LCSCs的Hu H7和原代HCC细胞Oct4和Sox2基因和蛋白表达,推测低浓度亚砷酸钠可能诱导LCSCs分化。
Objective To observe the response of Hep G2, Hep3B, Hu H7 and SMCC-7721 cells to sodium arsenite, the expression of CD133 and CD13 and the formation of tumor spheres. To investigate the effects of low concentrations of sodium arsenite on Hu H7 and primary HCC The changes of Oct4, Sox2 expression in cells were observed to investigate the mechanism of HAs inhibition by low concentration sodium arsenite. Methods The growth of Hep G2, Hep3B, Hu H7 and SMCC-7721 cells treated with 0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 μg / mL of different concentrations of sodium arsenite for 48, 72 and 96 h The expression of CD13 and CD133 in Hep G2, Hep3B, Hu H7, SMCC-7721 and primary HCC cells were detected by flow cytometry and the ability of tumor formation was analyzed to determine whether they contained hepatoma stem cells (LCSCs). The effects of 0.5 μg / mL sodium arsenite The changes of Oct4 and Sox2 gene and protein expression before and after treatment of Hu H7 and primary HCC cells were analyzed, and the effect of low concentration sodium arsenite on LCSCs was analyzed. Results Sodium arsenite treatment of HCC cells 48,72,96 h, arsenite significantly inhibited Hep G2, Hep3B, Hu H7 and SMCC-7721 cell growth inhibition of HCC cell proliferation with concentration and duration of action. Sodium arsenite treatment of HCC cells 96 h, 0.2,0.4μg / m L sodium arsenite has been significantly inhibited the growth of SMCC-7721 cells (P <0.05, P <0.01). 0.4μg / mL sodium arsenite significantly inhibited Hep G2 (P <0.01) and Hep3B (P <0.01) cell proliferation. 0.1, 0.2, 0.4 and 0.6 μg / mL sodium arsenite stimulated the proliferation of Hu H7 cells, but the difference was not statistically significant (P> 0.05). 0.8 μg / mL sodium arsenite significantly inhibited the proliferation of Hu H7 cells P <0.01). Sodium arsenite had a two-dimensional effect on the proliferation of Hu H7 cells at a low concentration in Hu H7 cells and the higher concentration of HuIFA 7 inhibited the proliferation of Hu H7 cells. With the extension of time, the inhibitory effect was enhanced at the same concentration. Low concentrations of sodium arsenite on SMCC-7721, Hep G2 and Hep3B cells almost no stimulation of proliferation. Hep G2, Hep3B and SMCC-7721 neither express CD133 nor form tumor spheres. Hu H7 and primary HCC cells express both CD133 and CD13 markers as well as tumor-forming ability. Hu H7 and primary HCC cells contain CD133 + CD13 + LCSCs. The expression of Oct4 and Sox2 genes and proteins in Hu H7 and primary HCC cells and 0.5 μg / mL sodium arsenite decreased the expression of Oct4 and Sox2 genes in Hu H7 and primary HCC cells. Conclusions Low concentrations of sodium arsenite downregulate or inhibit the expression of Oct4 and Sox2 genes in Hu H7 cells containing CD133 + CD13 + LCSCs and primary HCC cells. It is speculated that low concentrations of sodium arsenite may induce the differentiation of LCSCs.