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目的:体外观察长程缺氧环境对结肠癌细胞SW480和HCT116增殖能力的影响并探讨其可能的作用机制。方法:缺氧培养结肠癌SW480和HCT116细胞14～28d,采用MTT法和平板克隆法检测缺氧条件下2种结肠癌细胞增殖能力的改变;激光共聚焦显微镜下观察缺氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)和Wnt/β-catenin信号通路关键因子β-catenin的表达;蛋白质印迹法检测HIF-1α和β-catenin、糖原合成酶激酶-3β(glycogen synthase kinase-3β,GSK-3β)、Ser9被磷酸化的GSK-3β[phospho-glycogen synthase kinase-3β(Ser9),pGSK-3β(Ser9)]以及增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)的表达。以常氧下培养的这2种细胞作为对照。结果:细胞生长曲线和平板克隆检测结果均显示,长程缺氧适应后SW480和HCT116细胞的增殖能力较常氧下明显增强(P<0.01);激光共聚焦显微镜下观察发现,HCT116细胞在缺氧条件下HIF-1α的表达部位发生改变;蛋白质印迹法检测发现,与常氧组比较长程缺氧组细胞中HIF-1α(P<0.05)、β-catenin(P<0.05)和pGSK-3β(Ser9)(P<0.01)蛋白的表达均上调,而总GSK-3β的表达无明显变化,PCNA在缺氧条件下表达上调(与常氧组比较,在SW480细胞中P<0.05,在HCT116细胞中P<0.01)。结论:缺氧适应可促进结肠癌细胞SW480和HCT116的增殖,这可能是HIF-1α与Wnt/β-catenin信号转导通路共同作用的结果。
OBJECTIVE: To observe the effect of long-range hypoxia on the proliferation of colon cancer cells SW480 and HCT116 and to explore its possible mechanism. Methods: The hypoxia culture of colon cancer SW480 and HCT116 cells for 14 ~ 28 days, MTT assay and plate cloning method were used to detect the proliferation of the two colon cancer cell lines under hypoxia. Laser scanning confocal microscopy was used to observe the changes of hypoxia inducible factor-1α The expression of HIF-1α, β-catenin and hypoxia-inducible factor-1α, HIF-1α and Wnt / β-catenin signaling pathway were detected by Western blotting. 3β, GSK-3β), and phosphorylated GSK-3β (Ser9), pGSK-3β (Ser9) and proliferating cell nuclear antigen (PCNA) The two kinds of cells cultured under normoxia served as a control. Results: The results of cell growth curve and plate clone assay showed that the proliferative ability of SW480 and HCT116 cells were significantly enhanced under long-term hypoxia (P <0.01). Confocal laser scanning microscopy showed that HCT116 cells were significantly inhibited in hypoxia (P <0.05), β-catenin (P <0.05) and pGSK-3β (P <0.05) in the long-term hypoxia group compared with normoxia group Ser9) (P <0.01), while the expression of total GSK-3βwas not significantly changed. The expression of PCNA was up-regulated under hypoxia (P <0.05 compared with normoxic group in SW480 cells and in HCT116 cells In P <0.01). Conclusion: Hypoxia adaptation can promote the proliferation of colon cancer cells SW480 and HCT116, which may be the result of HIF-1α interaction with Wnt / β-catenin signal transduction pathway.