摘要目的 探讨PI3K/AKT信号通路对肝癌细胞MHCC-97H中CD90+干细胞比例及干细胞特性的影响. 方法 流式细胞术检测MHCC-97H细胞中流式抗体标记的CD90+细胞含量.PI3K/AKT通路抑制剂LY294002处理MHCC-97H细胞后,用流式细胞术分析MHCC-97H细胞中的CD90+干样细胞含量的变化,Western blot检测其相关蛋白的表达.平板克隆形成实验检测LY294002对MHCC-97H细胞中CD90+亚群和CD90-亚群克隆能力的影响.比较不同浓度梯度的CD90+亚群和CD90-亚群在裸鼠皮下成瘤的能力,以及加入LY294002后成瘤能力的变化.免疫组织化学检测CD90+亚群和CD90-亚群形成的瘤体中干细胞相关抗体CD90、SHP2及AKT和磷酸化-AKT (p-AKT)蛋白在加入抑制剂前后分别的变化情况.对数据进行t检验. 结果 MHCC-97H细胞中存在CD90+肝癌干样细胞,经过LY294002处理后,肝癌干样细胞比例由2.98％±0.08％下降为0.78％±0.08％(t=33.680,P＜0.01).LY294002可以显著降低CD90+亚群的克隆能力,用药前后分别为95.13％±3.78％和61.82％±7.23％(t=7.617,P＜0.01);对CD90-亚群的克隆能力影响不明显.LY294002能降低CD90+亚群的成瘤能力和相关肝癌干细胞CD90、SHP2和p-AKT蛋白的表达水平,但对AKT蛋白表达水平影响不大;对CD90亚群影响不明显. 结论 MHCC-97H细胞中的CD90+细胞具有干细胞特性,且依赖PI3K/AKT信号通路.

abstractsObjective To investigate the influence of the PI3K/AKT signaling pathway on the proportion and characteristics of the stem-like CD90+ subpopulation of the human hepatocellular carcinoma (HCC) cell line MHCC-97.Methods MHCC-97H cultures were treated with the PI3K/AKT pathway inhibitor LY294002.The proportion of the CD90+ subpopulation was determined by flow cytometry,and the expression of related proteins was measured by Westem blot.The clonogenicity of CD90+ and CD90-cells was measured by plate colony formation assay.The tumorigenicity was compared between CD90+ and CD90 subpopulations (with different concentrations) in xenograft experiments in nude mice,and the changes in tumorigenicity after the addition of LY294002 were evaluated.The changes in the expression of CD90,SHP2,P-AKT,and AKT in CD90+ and CD90-cell xenografts after the addition of LY294002 were examined.Data were analyzed using t test.Results LY294002 was capable of reducing the proportion of CD90+ HCC stem cells from 2.98％±0.08％ to 0.78％±0.08％ (t =32.400,P ＜ 0.01) and reducing the clonogenicity of CD90+ subpopulation from 95.13%±3.78％ to 61.82％±7.23％ (t =7.617,P ＜ 0.01).However,it showed no significant effect on the clonogenicity of CD90-subpopulation.LY294002 also reduced the tumorigenicity of CD90+ subpopulation and the expression of CD90,SHP2,and P-AKT in related HCC stem cells,but it did not significantly affect the expression of AKT.LY294002 had no significant inhibitory effect on the tumorigenicity of CD90-cells.Conclusion The CD90+ subpopulation of MHCC-97H cells has the characteristics of stem cells and is dependent on the PI3K/AKT signaling pathway.