缺氧诱导肝癌细胞获得干细胞特性的初步实验研究

摘要

Objective:To explore whether cancer stemness properties could be induced under hypoxia from hepatocellular carcinoma(HCC)cells in vitro.Methods:HepG2,SMMC-7721 cell lines were used to establish the model of hypoxia,and their morphological changes were observed by inverted microscope.Colony-forming ability was detected by colony formation assay.Tumor cell self-renewal capacity was analyzed by tumorsphere formation assay.The markers of stemness,including Oct-4,Sox-2 and Nanog,were detected by by real-time PCR and Western blot.Flow cytometry was used to detect the proportion of CD133+ cells.Results:Under hypoxic conditions,colony forming efficiency of HepG2 and SMMC-7721 cells were significantly higher than that of normoxia group(P<0.05).The tumorsphere formation rate of HepG2 and SMMC-7721 cells in hypoxia was significantly higher than that in normoxia(P<0.05).The expression of stemness related genes(Oct-4,Sox-2,Nanog)were low under normoxic condition,after treated by hypoxia for 48 h,their expression was increased significantly(P<0.05).The positive expression rate of CD133 in HepG2 and SMMC-7721 cells was significantly increased under hypoxia(P<0.05).The expression of CD133 protein was significantly increased under hypoxia for 36 h and 48 h(P<0.05).Conclusion:Hypoxia induces the formation of cancer stemness from HCC cells.%目的:观察缺氧对肝癌干细胞特性的影响.方法:用肝癌细胞系HepG2、SMMC-7721建立细胞缺氧培养模型,利用倒置显微镜观察细胞形态学变化;克隆形成实验检测肿瘤细胞克隆形成能力;"肿瘤球"形成实验检测肿瘤细胞自我更新能力;实时定量RT-PCR和Western blot检测干细胞相关基因Oct-4、Sox-2、Nanog mRNA和蛋白表达;采用流式细胞术检测干细胞表面标志物CD133表达.结果:缺氧条件下HepG2、SMMC-7721细胞克隆形成率显著高于常氧组(P<0.05).缺氧条件下HepG2、SMMC-7721细胞肿瘤球形成率显著高于常氧组(P<0.05).常氧条件下HepG2、SMMC-7721细胞中干细胞相关基因Oct-4、Sox-2、Nanog mRNA及蛋白表达水平极低,缺氧处理48 h后,其mRNA及蛋白表达水平明显增高(P<0.05).缺氧条件下HepG2、SMMC-7721细胞CD133阳性表达率表达量显著增加(P<0.05).在缺氧36 h和48 h,CD133蛋白表达水平显著增强(P<0.05).结论:缺氧能诱导肝癌细胞干细胞特性的获得.