银杏内脂B对人骨肉瘤Saos-2细胞的抑制作用研究

Inhibitory effect of ginkgolide B on human osteosarcoma Saos-2 cells

目的探讨银杏内脂B(GB)对骨肉瘤(OS)细胞增殖、凋亡和迁移侵袭的影响以及潜在机制。方法将人骨肉瘤Saos-2细胞分别在含300、600和1200μmol/L GB的培养液中培养24、48和72 h,采用CCK-8法检测Saos-2细胞的增殖情况,流式细胞仪检测Saos-2细胞凋亡率,Transwell小室迁移与侵袭实验检测GB对Saos-2细胞迁移和侵袭能力的影响;提取GB处理后Saos-2细胞的mRNA和蛋白,采用实时荧光定量PCR和Western blotting检测Bcl-2、Bax、MMP-2和MMP-9的基因及蛋白表达水平,Western blotting法检测Erk和Akt磷酸化及cleaved-caspase-3水平。结果 GB能明显抑制Saos-2细胞的增殖,且呈时间和浓度依赖性,600和1200μmol/L GB可诱导Saos-2细胞凋亡,GB对Saos-2细胞的迁移及侵袭的抑制作用呈浓度依赖性;GB可升高Bax mRNA和蛋白水平,但降低Bcl-2、MMP-2和MMP-9的mRNA和蛋白水平;高浓度GB能抑制Erk和Akt的磷酸化且升高cleaved-caspase-3水平。结论 GB具有抑制Saos-2细胞增殖及迁移侵袭和诱导细胞凋亡作用,其机制可能与抑制Erk和Akt磷酸化有关。

Objective To explore the effect of ginkgolide B（GB）on the proliferation, apoptosis, migration and invasion of os- teosarcoma（OS）cells as well as the potential mechanisms. Methods Human OS Saos-2 cells were incubated with 300, 600 and 1200 μmol/L GB for 24, 48 and 72 h. We detected cells viability, apoptosis, migration and invasion with cell counting kit-8（ CCK-8）, flow cytometry and Transwell assay, separately. Using the extraction of the mRNA and protein of Saos-2 cells pretreated with GB, we detected the mRNA and protein levels of Bcl-2, Bax, caspase-3, MMP-2 and MMP-9 by using quantitative real-time PCR and Western blotting. The phosphorylation of Erk and Akt and level of cleaved-caspase-3 were detected by Western blotting. Results GB significantly inhibited the proliferation of Saos-2 cells in a time-and dose-dependent manner. GB at 600 and 1200 μmol/L can induce the apoptosis of OS cells. GB exerted a dose-dependent role in the suppression of migration and invasion of OS cells. GB could increase the mRNA and protein lev- els of Bax, but decreased the mRNA and protein levels of Bcl-2, MMP-2 and MMP-9. The high concentration of GB could inhibit the phosphorylation of Erk and Akt and increase the level of cleaved-caspase-3. Conclusion GB could inhibit the proliferation, migration and invasion of Saos-2 ceils and induce apoptosis, which may be related to the inhibition of Erk and Akt phosphorylation.