低氧诱导对人肝癌SMMC7721细胞生物学行为的影响

Influence of Hypoxia on Hepatoma Carcinoma Cell Line SMMC7721 in Vitro

目的研究低氧对体外培养的人肝癌细胞株SMMC7721增殖、细胞周期、凋亡和黏附、迁移能力的影响,探讨其中可能存在的分子机制.方法通过氯化钴诱导人肝癌SMMC7721细胞低氧,MTT法、流式细胞术、细胞黏附试验、transwell小室迁移实验观察低氧对肝癌细胞增殖、凋亡和细胞周期以及细胞黏附和迁移能力的影响;采用RT-PCR和免疫细胞化学方法检测低氧对细胞缺氧诱导因子-1α(HIF-1α)、血管内皮生长因子(VEGF)、基质金属蛋白酶2(MMP2)mRNA和蛋白表达的影响.结果 MTT法显示氯化钴诱导的低氧对肝癌细胞生长起抑制作用;低氧24 h,流式细胞术显示细胞凋亡、坏死增加,细胞周期G0/G1期、S期细胞比例减少,G2/M期比例增加;黏附试验显示低氧后细胞黏附力增强(P<0.01),transwell小室实验显示,低氧后细胞迁移能力较常氧时明显增加(P<0.05);RT-PCR和细胞免疫化学检测显示低氧条件下HIF-1α、VEGF、MMP2的mRNA和蛋白表达均较常氧时增加(P<0.05).结论氯化钴诱导低氧可抑制SMMC7721细胞增殖,使细胞周期阻滞于G2/M期,细胞凋亡、坏死增加,同时细胞黏附、迁移能力增强;低氧时HIF-1αmRNA和蛋白表达增加,并通过调节及其下游靶基因VEGF、MMP2的表达参与了这一生物学行为的转变.

Objective To investigate the effect of hypoxia on human hepatoma carcinoma cell line SMMC7721 cultured in vitro.and to explore the possible molecular mechanism.Methods The chemical method using COCl2 to induce hypoxia environment of SMMC7721 cell was performed.The effect of hypoxia on cell growth,cycle and apoptosis,and adhesion and migration ability were detected by MTT assay,flow cytometry,cell adhesion test,and transwell chamber methods,respectively.RT-PCR and immunocytochemistry methods were used to detect HIF-1α,VEGF,MMP2 mRNA and protein expression.Results MTT assay showed that cell growth was inhibited under hypoxia induced by COCl2.Flow cytometry showed that cell apoptosis,necrosis rate were increased,G0/G1 cell cycle phase,S phase cells decreased,G2/M phase was increased flow cytometry showed that cell apoptosis,apoptosis and necrosis rate were increased,G0/G1 phase and S phase cells decreased,G2/M phase cells were increased 24 h after hypoxia.Adhesion test showed cell adhesion was enhanced 24 h after hypoxia（P0.01）,transwell chamber experiments showed that the cell migration significantly increased 24 h after hypoxia compared with normoxia（P0.05）.RT-PCR and immunocytochemical staining showed that mRNA and protein expression of HIF-1α,VEGF,MMP2 increased under hypoxic conditions compared with normoxia（P0.05）.Conclusion The proliferation of SMMC7721 cell can be inhibited by hypoxia,resulting in the restriction of SMMC7721 cell in its G2/M phase of the cell cycle and enhance apoptosis and necrosis.HIF-1α and its downstream target genes VEGF and MMP2 mRNA and protein expression are involved in the biological behavior changes.