氧化苦参碱对骨肉瘤U2OS细胞增殖的抑制作用及其机制研究

Inhibitory Effect of Oxymatrine on the Proliferation of Osteosarcoma U2OS Cell and Its Mechanism

目的:在分子生物学水平上探讨氧化苦参碱(Oxymatrine,OMT)对骨肉瘤U2OS细胞的生长抑制作用和潜在的作用机制。方法:体外培养骨肉瘤U2OS细胞,用不同浓度梯度的氧化苦参碱处理细胞,光学显微镜观察骨肉瘤细胞形态的变化;四甲基偶氮唑盐(MTT)法检测不同浓度氧化苦参碱对U2OS细胞增殖的影响;AnnexinⅤ-FITC/PI荧光双标流式细胞仪分析OMT处理后的U2OS细胞周期阻滞和凋亡的变化;RT-PCR方法分析凋亡相关分子(p53,bax,Bcl-2)mRNA的表达水平。结果:MTT法结果表明氧化苦参碱能够抑制U2OS细胞的增殖并呈剂量依赖性;光学显微镜下观察到氧化苦参碱处理后的U2OS细胞具有凋亡特征的形态学变化;流式细胞仪检测结果表明氧化苦参碱处理48h后,细胞凋亡率与药物浓度呈正相关;氧化苦参碱还能够诱导细胞周期阻滞于G0/G1期,并呈现浓度依赖性;RT-PCR检测结果表明氧化苦参碱处理后,U2OS细胞中p53mRNA和bax mRNA的表达水平明显上升,Bcl-2mRNA表达水平明显下降,并呈现一定的剂量依赖关系。结论:氧化苦参碱能够明显抑制骨肉瘤U2OS细胞的增殖,导致细胞周期阻滞并诱导细胞凋亡,具体分子机制可能和p53,Bcl-2,bax等凋亡调控基因相关。

Objective:To investigate the inhibitory effect of oxymatrine(OMT)on the growth of osteosarcoma U2 OS cell and its potential mechanism.Methods:U2 OS cell of osteosarcoma was cultured in vitro and treated with oxymatrine at different concentrations.The morphological change of osteosarcoma cell was observed under light microscope.The effect of oxymatrine on U2 OS cell proliferation was examined by MTT assay.The flow cytometry using AnnexinⅤ-FITC/PI staining was employed to measure the cell cycle and cell apoptosis of U2 OS.RT-PCR was used to detect mRNA expression levels of p53,bax and Bcl-2.Results:MTT assay showed that oxymatrine could inhibit the proliferation of U2 OS cell in a dose-dependent manner.The morphological change of apoptosis in U2 OS cell treated with oxymatrine was observed under light microscope.The result of flow cytometry showed that there was a positive correlation between the apoptosis rate and the concentration of oxymatrine after 48 htreatment.Oxymatrine also induced cell cycle arrest at G0/G1 phase,and it was concentration-dependent.The result of RT-PCR showed that the expression of p53 mRNA and bax mRNA in U2 OS cell increased obviously,while the expression of Bcl-2 mRNA decreased obviously,and there was a dose-dependent relationship after oxymatrine treatment.Conclusion:Oxymatrine can inhibit the proliferation of osteosarcoma U2 OS cell through induction of cell cycle arrest and apoptosis.The detailed molecular mechanism may be related to p53,Bcl-2,bax and other apoptosis-regulating genes.