粒细胞-巨噬细胞集落刺激因子对结肠癌肝转移的促进作用及其可能机制

Promoting effect of granulocyte-macrophage colony stimulating factor on liver metastasis of colon cancer and its underlying mechanisms

目的:探讨粒细胞-巨噬细胞集落因子(granulocyte-macrophage colony stimulating factor,GM-CSF)对结肠癌肝转移的促进作用及其可能机制。方法:采用稳定转染方法将GM-CSF基因以及靶向GM-CSF的sh RNA分别导入人结肠癌细胞系HCT116和SW480,用ELISA法检测GM-CSF表达水平。脾内注射结肠癌细胞建立结肠癌肝转移模型,部分模型小鼠隔天腹腔注射不同剂量(3、0.6μg)的重组人GM-CSF。模型建立后4周后观察肝大体及肿瘤转移灶,实时荧光定量PCR检测神经钙黏素(Ncadherin)和基质金属蛋白酶2(matrix metalloproteinase 2,MMP2)的表达,Western blotting检测上皮钙黏素(E-cadherin)的表达。结果:根据GM-CSF表达量建立GM-CSF低、中、高表达细胞系(GMlo、GMint、GMhiHCT116)和GMKDSW480细胞系。大体和病理结果均显示,接种高表达GM-CSF的HCT116细胞或外源性给予GM-CSF明显增加小鼠结肠癌肝转移灶数量,而敲低GM-CSF的作用则相反。GM-CSF能够显著上调N-cadherin和MMP2的表达,同时下调E-cadherin的表达(P<0.05)。结论:GM-CSF能够促进结肠癌移植瘤的肝转移,该效应可能与E-cadherin表达的下调和N-cadherin以及MMP2表达的上调有关。

Objective： To investigate the effect of granuloeyte-macrophage colony stimulating factor（GM-CSF） on liver metastasis of colon cancer and the underlying mechanisms. Methods： GM-CSF gene and shRNA targeting GM-CSF were stably transfected into hu- man colon cancer HCT 116 and SW480 cell lines, respectively; the expression of GM-CSF was detected by ELISA. Liver metastasis model of colon cancer was established by injecting cancer cells into spleen. Some of the model mice were intraperitoneally injected with recombinant human GM-CSF every other day （3 gg, 0.6 gg/mice）. Four weeks after model establishment, liver gross and tumor metastasis were observed. The expression of N-cadherin and matrix metalloproteinase 2 （MMP2） was detected by Real-time fluorescent quantitative PCR while E-cadherin was detected by Western blotting. Results： According the expression of GM-CSF, HCTI 16 cells were grouped into low, median and high expression subgroups （GM--, GM--t, GM-HCT116）; Meanwhile, GM-DSW480 group （GM-CSF knockdown） was established. Liver gross and pathological results showed that injecting of GM-CSF-overexpressed HCT116 cells or exogenous GM-CSF remarkably increased the amounts of liver metastasis; In contrast, knockdown of GM-CSF showed the opposite effect. Furthermore, GM-CSF over-expression significantly up-regulated the levels of N-cadherin and MMP2 as well as down-regulated the levels of E-cadherin （P〈0.05）. Conclusion： GM-CSF can potently promote liver metastasis of colon cancer xenograft, which may be due to the down-regulation of E-cadherin and up-regulation of N-cadherin and MMP2.