奇果菌素诱导骨肉瘤U2OS细胞凋亡及其机制的研究

目的 研究奇果菌素（grifolin）诱导骨肉瘤U2OS细胞体外生长抑制及诱导凋亡的作用及其机制.方法 应用四甲基偶氮唑盐（MTT）比色法测定奇果菌素对U2OS细胞的生长抑制实验;荧光显微镜下（Hoechst 33258荧光染色）观察细胞核形态学变化;流式细胞仪检测细胞的凋亡率;Western blot法检测Akt、GSK3、FKHRL蛋白表达.结果 奇果菌素的浓度在50μm以上时对骨肉瘤U2OS细胞具有明显的生长抑制作用,呈明显的时间和浓度效应关系;并能诱导细胞发生凋亡,出现凋亡小体,呈典型的凋亡细胞形态;随着药物浓度的增加和作用时间的延长,细胞的生长抑制率及细胞凋亡率均明显升高,50.0μm的奇果菌素作用U2OS细胞0,6,12,24h后,细胞出现不同程度的亚G1峰,细胞凋亡率分别为1.96%、13.91%、52.6%、63.5%;Western blot法分析表明U2OS细胞经过50.0μm和100μm的奇果菌素作用24h后,U2OS细胞的Akt、FKHRL、GSK3的表达均下调,表示其表达水平及活性显著降低.结论 奇果菌素能够通过诱导人骨肉瘤U2OS细胞发生凋亡而发挥抑制U2OS细胞生长作用,尤其当奇果菌素的浓度＞50.0μm,其对U2OS细胞增生的抑制作用呈剂量和时间依赖性,抑制Akt和GSK3的活性是奇果菌素体外诱导人骨肉瘤U2OS细胞发生凋亡的重要作用机制之一.

A study of apoptosis of human osteosarcoma U2OS cells induced by grifolin and its mechanism

Objective To study the effect and mechanism of the vitro inhibition of growth and induction of apoptosis in human osteosarcoma U2OS cells induced by grifolin. Methods The experiment of inhibiting the growth of human osteosarcoma U2OS cells by grifolin was assessed by the 3-（4,5）-dimethylthiahiazo （-z-yl）-3,5-diphenytetrazoliumromide （MTT） colorimetric assay. The morphological changes of the cell nucleus were monitored under fluorescence microscope （Hoechst 33258 staining）. The apoptosis rate was detected by flow cytometry. The protein expressions ofAkt, GSK3 and FKHRL were tested by Western blot analysis. Results Grifolin suppressed the proliferation of human osteosarcoma U2OS cells distinctly, when its concentration was over 50lun. The suppression was obviously in a dose- and time- dependent manner. In addition, grifolin was able to induce apoptosis. There were apoptotic bodies which presented a typical form of apoptotic cells. Along with the increase of the concentration and the extension of the action time, both the growth inhibition rate and apoptosis rate increased significantly. After 50.0pro grifolin acted on human osteosarcoma U2OS cells for 0, 6, 12 and 24 hours respectively, varying degrees of sub-G1 peaks appeared in cells and the apoptosis rates were 1.96%, 13.91%, 52.6% and 63.5% accordingly. Western blot analysis indicated that after 50.0~trn and 100pro grifolin acted on human osteosarcoma U2OS cells for 24 hours, the expressions ofAkt, FKHRL and GSK3 in U2OS cells all fell, which showed that the expression levels and activity decreased apparently. Conclusions Grifolin can inhibit the growth of human osteosarcoma U2OS cells by inducing their apoptosis, especially when its concentration is more than 50.0μm. The suppression on the proliferation of U2OS cells is in a dose- and time- dependent manner. Inhibiting the activity of Akt and GSK3 is one of the important mechanisms of apoptosis of human osteosarcoma U2OS cells induced by grifolin in vitro.