目的： 研究氯化钴（CoCl2）模拟的细胞体外缺氧微环境对结直肠癌细胞株SW480及Caco-2上皮间质转化及DNA同源重组修复的影响并探究其机制。方法：应用不同浓度的氯化钴（CoCl2）处理SW480及Caco2细胞72 h后，CCK8法检测细胞增殖能力，划痕及Transwell试验检测细胞的迁移及侵袭能力，流式细胞术（FCM）检测细胞周期及凋亡情况，RT-PCR及Western blotting实验检测细胞内相关基因mRNA及蛋白水平的变化情况。结果： CCK-8实验提示细胞在缺氧后增殖能力明显增加（P<005）；划痕试验及Transwell侵袭实验提示细胞在缺氧条件下迁移及侵袭能力明显增强（P<0.05）；流式细胞术提示细胞在缺氧后被阻滞在S期（P<0.05），凋亡率明显下降（P<0.05）；RT-PCR试验表明缺氧后细胞内缺氧诱导因子1-α（hypoxia inducible factor 1 alpha，HIF-1α）及RAD51的mRNA水平上调（P<0.05）；Western blotting实验表明在缺氧环境下细胞内HIF-1α表达上调（P<0.05），上皮-间质转化（epithelial-mesenchymal transition）相关蛋白：钙黏附蛋白E（E-cadherin）表达下调，波形蛋白（vimentin）及转录抑制因子（snail）表达上调（P<0.05），DNA同源重组相关蛋白BRCA1及RAD51表达上调（P<0.05），PI3K/AKT（磷脂酰肌醇-3-羟激酶）信号通路下游关键信号分子AKT1及共济失调毛细血管扩张症突变基因(ataxia-telangiectasia mutant gene)的编码产物ATM激酶磷酸化水平显著上调（P<0.05）。结论：慢性缺氧环境可促进结直肠癌细胞SW480及Caco-2的增殖、迁移和侵袭能力，抑制凋亡，其机制可能与HIF-1α/PI3K-AKT、HIF1-α/ATM信号通路介导EMT及DNA同源重组修复过程有关。

Objective:To investigate effect of hypoxic microenviroment in vitro which built by cobalt chloride (CoCl2) on epithelial mesenchymal transition and DNA homologous recombination repair of colorectal cancer SW480 and Caco-2 line cells and to explore their mechanisms. Methods: After the SW480 and Caco-2 cells were treated with various concentrations of CoCl2 for 72 h, proliferation abilities of the cells were detected by CCK8 assay, migration and invasion abilities of the cells detected by scratch test and transwell assay, cell cycles and apoptosis status of the cells detected by flow cytometry (FCM), changes of mRNA and protein levels of associated gene in the cells detected by RT-PCR and Western blotting assays. Results: CCK8 assay prompted that proliferation ability of the cells significantly increased after hypoxia (P<0.05); scratch and transwell tests suggested that under hypoxia, migration and invasion abilities of the cells obviously enhanced (P<0.05); FCM disclosed that after anoxic treatment the cells were maintained at S phage and apoptosis rates of the cells significantly decreased (P<0.05); RT-PCR assay showed that mRNA levels of HIF-1α and RAD51 in the cells after hypoxic treatment raised (P<0.05); Western blotting assay indicated that under hypoxic microenviroment in the cells expression of HIF1-α enhanced (P<0.05), expression of epithelial mesenchymal transition (EMT) associated proteins, namely E-cadherin was down-regulated, expressions of vimentin and transcription inhibitor (snail) were up-regulated (P<0.05), expressions of DNA homologous recombination associated protein BRCA1 and RAD51 were up-regulated (P<0.05), phosphorylation levels of down stream key molecule Akt1 on PI3K/AKT (phosphatidyl inositol-3-hydroxy kinase) signaling pathway and ATM kinase encoded by ataxia-telangiectasia mutant gene were significantly enhanced (P<0.05). Conclusion: Chronic hypoxia enviroment could promote abilities of proliferation, migration and invasion of the colorectal cancer SW480 and Caco-2 cells as well as inhibit apoptosis of the cells. Mechanism of the actions might related to HIF-1α/PI3K and HIF-1α/ATM signaling pathways mediated EMT and DNA homologous recombination repair process.

重庆市科委自然科学基金项目（No.cstc2012jjA10038）