中国普外基础与临床杂志

中国普外基础与临床杂志

乏氧环境下 HIF-1α 对肝癌细胞逆向分化影响的实验研究

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目的 探讨体外乏氧环境下低氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)对诱导肝癌细胞逆向分化为肝癌干细胞并维持恶性生物学行为的影响。 方法 采用免疫磁珠分选出 HepG2 细胞中的 CD133 阴性细胞,分为 2 个大组:转染组转染 siRNA-HIF-1α 以沉默 HIF-1α 基因的表达,空白对照组不转染任何 siRNA 片段。2 类细胞分别进行常氧及乏氧条件培养,本实验共计 4 组。采用 MTT、克隆形成实验及 Trans-well 小室实验检测细胞的增殖和侵袭能力,采用 Western blot 法及 RT-PCR 法检测细胞中 HIF-1α、CD133、CD90 及 CD44 mRNA 及其蛋白的表达。 结果 MTT 实验结果显示:4 组细胞的殖率随乏氧时间延长而增高;24 h 及以后,与空白对照组相比,经 siRNA-HIF-1α 转染后,转染常氧组和乏氧组的细胞增殖率降低(P<0.05)。平板克隆实验结果显示:转染常氧组与转染乏氧组、空白对照常氧组与空白对照乏氧组比较差异均有统计学意义(P<0.05)。Transwell 小室实验结果显示:乏氧培养后,转染组与空白对照组相比,迁移至下室的细胞数目减少(P<0.05)。Western blot 及 RT-PCR 结果显示:空白对照乏氧组中 HIF-1α 及肿瘤干细胞标志物(CD133、CD90、CD44)蛋白及其 mRNA 的表达水平均高于其余 3 组(P<0.05);经 siRNA-HIF-1α 转染后,转染乏氧组中 HIF-1α 及肿瘤干细胞标志物(CD133、CD90、CD44)蛋白及 mRNA 的表达水平均较转染常氧组和空白对照常氧组降低(P<0.05)。 结论 在乏氧环境下,低氧诱导因子 HIF-1α 可促进肝癌细胞逆向分化为肝癌干细胞并增强其恶性生物学行为。

Objective To explore the effects of hypoxia inducible factor-1 alpha (HIF-1α) on the reverse differentiation ofhepatocellular carcinoma cells into liver cancer stem cells, and the maintenance of malignant biological behavior in hypoxic environment. Methods CD133-negative cells in HepG2 cells were separated by immunomagnetic beads and divided into two groups. The cells of siRNA group were transfected with siRNA-HIF-1α to silence the expression of HIF-1α gene, while cells of the blank control group did not transfect any siRNA fragments. Two groups of cells were cultured under normal and hypoxic conditions respectively. MTT, cloning and trans-well chamber experiments were used to detect the proliferation and invasion ability of cells. Western blot and real-time PCR (RT-PCR) were used to detect the expression of HIF-1α, CD133, CD90, and CD44 protein and mRNA in cells. Results MTT results showed that the cell proliferation rate increased with the prolongation of hypoxia in four groups. Compared with the control group at 24, 32, 40, and 48 hours, the cell proliferation rate decreased significantly after siRNA-HIF-1a transfection, on both two kinds of cultured conditions (P<0.05). The results of plate cloning experiment showed that the number of cell-forming clones increased significantly after hypoxic culture (there were significant differences between transfected hypoxic group and transfected hypoxic group, blank control hypoxic group and blank control hypoxic group,P<0.05); and the formation of transfected hypoxic condition group at the same time of hypoxia was also significant (P<0.05). The number of clones were significantly less than that of the blank control group at the hypoxic condition (P<0.05). Transwell lab experiment showed that after hypoxic culture, the number of cells migrated to the inferior chamber in transfection group was significantly reduced compared with that of blank control group (P<0.05). Western blot and RT-PCR results showed that the expression levels of HIF-1α protein and tumor stem cell markers (CD133, CD90, and CD44 protein) in the blank control hypoxic condition group were significantly higher than those in the other three groups (P<0.05); after siRNA-HIF-1a transfection, HIF-1α mRNA and tumor stem cell markers mRNA (CD133, CD90, and CD44 mRNA) in the transfected hypoxic condition group were significantly lower than those in the transfected normal condition group and blank control normal condition group (P<0.05). Conclusions In hypoxia environment, hypoxia inducible factor HIF-1 alpha (HIF-1α) can promote hepatocellular carcinoma cells to differentiate into liver cancer stem cells and enhance their malignant biological behavior.

关键词: 人肝癌HepG2细胞; 肝癌干细胞; 乏氧环境; 低氧诱导因子-1α; 逆分化

Key words: hepatocellular carcinoma HepG2 cells; liver cancer stem cells; hypoxia; hypoxia inducible factor-1 alpha; reverse differentiation

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