右美托咪定对缺氧诱导人肝癌细胞增殖和血管生成的影响

Effect of Dexmedetomidine on Proliferation and Angiogenesis of Human Hepatocellular Carcinoma Cell Cultured in Hypoxia Condition and Possible Mechanism

【目的】通过建立肝癌细胞体外缺氧模型分析右美托咪定对缺氧诱导肝癌细胞作用效应的影响及其初步机制的探讨。【方法】人肝癌细胞株MHCC97H、SMCC7721分别使用化学(二氯化钴)及物理(1%O_2)这两种方法来建立缺氧状态下的肝癌细胞体外模型,均培养在含有或者没有100μmol/L的右美托咪定的培养基中。将以上处理的细胞进行细胞增殖实验、单克隆形成实验、小管形成实验来检测右美托咪定对缺氧条件培养的肝癌细胞的增殖能力及血管生成能力的影响,并同时使用western blot检测α2A、HIF-1a及VEGF蛋白的表达情况。【结果】化学方法诱导的肝癌细胞缺氧可以加快肝癌细胞株MHCC97H、SMCC7721的增殖速度(二氯化钴组vs.对照组,MHCC97H和SMCC7721增殖效应分别为:第3天,142.2%和133.8%;第4天,134.7%和131.0%;第5天,133.5%和136.2%,均P<0.05),也诱导肝癌细胞的血管生成能力加强。而右美托咪定可抑制缺氧引起的肝癌细胞的增殖效应(联合组vs.二氯化钴组,MHCC97H和SMCC7721增殖效应分别为:第3天,55.7%vs 60.7%;第4天,46.9%vs 58.1%;第5天,46.4%vs 57.0%,均P<0.05),减弱肝癌细胞的血管生成能力。而使用物理方法建立的肝癌细胞体外缺氧模型同样得到一致的结果。另外Western blot结果发现右美托咪定可以下调缺氧诱导的肝癌细胞株MHCC97H、SMCC7721的α2A、HIF-1a及VEGF等蛋白的表达。【结论】缺氧可以加快肝癌细胞株MHCC97H、SMCC7721的增殖,同时诱导血管生成能力加强,右美托咪定则可能通过激活α2A肾上腺受体,下调HIF-1a及VEGF蛋白的表达来抑制缺氧诱导的肝癌细胞的作用效应。

【Objective】This study was conducted to examine the effects of dexmedetomidine on the proliferation and angiogenesis of MHCC97 H and SMCC7721 human hepatocellular carcinoma（HCC）cell lines cultured in hypoxia condition in vitro,and investigated the possible mechanism involved.【Methods】MHCC97H and SMCC7721 human HCC cell lines under hypoxia culture condition were treated with presence or absence of dexmedetomidine（100 μmol/L）.Cell viability,colony formation,vasculogenic mimicry（VM）formation were assessed.The effects of dexmedetomidine on α-2A adrenergic receptor（α2A）,hypoxia induced factor-1a（HIF-1a）,and vascular endothelial growth factor（VEGF）protein expression were evaluated with Western blot analysis.【Results】Cell proliferation assay and colony formation assay indicated that hypoxia obviously promoted the proliferation of MHCC97 H and SMCC7721 cells（CoCl_2 group vs corresponding control group,the proliferation rate of MHCC97 H and SMCC7721：Day 3,142.2%and 133.8 %;Day 4,134.7 % and 131.0 %;Day 5,133.5 % and 136.2 %;all P〈0.05）,and VM formation assay suggested that hypoxia increased angiogenesis of MHCC97 H and SMCC7721 cells.Whereas dexmedetomidine significantly inhibited the proliferation（Dex＋CoCl_2 group vs CoCl_2 group,the proliferation rate of MHCC97 H and SMCC7721：Day 3,55.7% vs 60.7%;Day 4,46.9% vs58.1%;Day 5,46.4% vs 57.0%,all P〈0.05）and angiogenesis of MHCC97 H,SMCC7721 cells induced by hypoxia.Dexmedetomidine may exert these functions by activating α-2A adrenergic receptor,causing an decrease in HIF-1a and VEGF protein,while hypoxia activated HIF-1a and VEGF protein to promote the growth and angiogenesis of cells.【Conclusion】The findings provide evidence that hypoxia could promote the proliferation and angiogenesis of MHCC97 H and SMCC7721 cells,while dexmedetomidine might inhibit these effects by down-regulating HIF-1a and VEGF protein expression through activating α-2A adrenergic receptor.