摘要
背景和目的:脱—γ—羧基凝血酶原(Des-γ-carboxy-prothrombin,DCP)是肝细胞肝癌产生的异常凝血酶原,其机制可能与肝癌细胞缺乏或对维生素K利用障碍,造成依赖维生素K的γ谷氨酰羧化酶及辅酶活性降低,导致内质网产生的凝血酶原N端10个谷氨酸残基羧化不全致DCP大量产生并分泌至血液中。我们认为,DCP不但可以作为肝细胞癌(hepatocellular carcinoma cells HCC)诊断的标志物,更重要的作用在于其可能具有刺激肝癌细胞增殖作用。所以,抑制肝癌细胞DCP的产生和降低血液中含量,可能达到抑制DCP对肝癌细胞的进一步刺激增殖作用,达到辅助治疗肝癌的目的。维生素K类药物及其衍生物被发现可能具有一定的抑制肝癌细胞产生DCP作用,降低血液DCP水平,有效抑制肝癌细胞生长,改善临床症状,作用机制可能与其促进肝癌细胞γ谷氨酰羧化酶活性有关。本研究通过实验观察了维生素K2对肝细胞肝癌的抑制作用,证实了其与降低DCP表达之间的相关性。实验方法:体外实验,以MTT法检测了VitK2对人肝癌细胞(HCC)生长抑制作用,以集落形成法测定VitK2对长时间接触肿瘤细胞克隆原细胞的抑制活性。以Transwell小室模型方法观察了VitK2对HCC细胞体外侵袭能力的抑制作用。体内实验,以裸鼠接种人肝癌细胞,灌胃给予VitK2,观察其对HCC细胞的生长抑制作用。DCP表达水平检测方法,以ELISA法检测体外培养细胞上清液和动物血液DCP水平;以Western blot和免疫组织化学法分析HCC细胞DCP表达状态。实验结果:体外结果显示,不同剂量VitK2与HCC细胞孵育不同时间,VitK2对HCC增殖具有一定的生长抑制作用,并呈现剂量和接触时间依赖性关系。VitK2具有明显抑制HCC细胞侵袭和穿过Matrigel的能力,其对肿瘤细胞的抑制增殖作用在裸鼠移植HCC细胞实验中得到证实。在研究VitK2抑制HCC细胞增殖与DCP表达水平相关性方面,VitK2(2-40μM)明显降低HCC细胞培养上清液中DCP表达水平,对HCC生长抑制作用和DCP水平下降呈现相关性,这种作用在裸鼠移植人肝癌细胞表达DCP及VitK2治疗实验中得到证实。结论:VitK2具有抑制人肝癌细胞增殖的作用,其机制与降低细胞表达DCP水平有关。
Background:Des-γ-carboxyl prothrombin(DCP) is a serum protein produced by hepatocellular carcinoma(HCC) cells in the absence of vitamin K.Serum and tissue DCP expressions are thought to reflect the biological malignant potential of HCC. Hence,we aimed to examine the efficacy of vitamin K2 on the production of DCP as well as tumor cell growth and invasion.Methods:Cell growth and viability were evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide(MTT) assay.The in vivo efficacy of vitamin K2 was examined in nude mice bearing HCC cells.A 24-well transwell chamber was used to evaluate the motility and invasive ability of HCC cells.Levels of DCP in supernatant of cultures and in serum of mice were measured using the electrochemiluminescence immunoassay method.Western blot and immunohistochemical analysis were employed to evaluate the expression of DCP in HCC cells.Results:Vitamin K2(2-40μM) significantly decreased the levels of DCP production in supernatant of PLC/PRF/5 and HepG-2 cells cultures and in serum of nude mice bearing HCC xenografts.Inhibition of DCP expression was also observed using the assays of Western blot analysis in HCC cultures and immunohistochemical analysis in HCC xenografts in mice.As a result of administration of vitamin K2,the capacity of HCC cells growth was inhibited and the invasion and migration of tumor cells were decreased.Furthermore,the inhibitory effects of HCC cells growth were also observed in vivo and the sensitivity was well correlated with the declines of DCP levels in serum of mice.Conclusion:Vitamin K2 might suppress the growth and invasion of hepatocellular carcinoma cells via decreasing the expression of DCP.
引文
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