短叶松素-3-乙酸酯对人神经胶质瘤U251细胞增殖和凋亡的影响

Effects of Pinobanksin-3-acetate on Proliferation and Apoptosis of Human Glioma U251 Cells

目的探讨短叶松素-3-乙酸酯(Pinobanksin-3-acetate,PB3A)对人神经胶质瘤U251细胞增殖、凋亡的影响及其作用机制。方法体外培养人神经胶质瘤U251细胞,将细胞分为空白对照组(0μg·mL^-1)和PB3A组(2.5、5、10、20、40、80μg·mL^-1)。以倒置显微镜观察PB3A处理后细胞的形态变化;CCK-8法检测细胞增殖抑制率;EDU实验检测细胞增殖能力;流式细胞术检测细胞周期和细胞凋亡率;Western Blot法检测Bcl-2、Bax、Caspase-3、CyclinB1及CyclinD1蛋白的表达。结果与空白对照组比较,PB3A在5~80μg·mL^-1范围内的细胞增殖抑制率均明显增高(P<0.05,P<0.01),且呈现出浓度和时间依赖性;PB3A 10、20、40μg·mL^-1组EDU阳性细胞率均明显降低(P<0.01),细胞凋亡率均明显升高(P<0.01),G0/G1期细胞比例均明显降低(P<0.05,P<0.01),G2/M期细胞比例均明显升高(P<0.05,P<0.01);PB3A 20、40μg·mL^-1组均能明显上调Bax、Caspase-3蛋白的表达(P<0.01),下调Bcl-2、CyclinB1蛋白的表达(P<0.01),对CyclinD1蛋白的表达均无明显影响。结论PB3A能抑制人神经胶质瘤U251细胞增殖,诱导细胞凋亡,并阻滞细胞周期于G2/M期,其机制可能与调控凋亡和周期相关蛋白的表达有关。

Objective To study the anti-proliferation and pro-apoptotic effects of Pinobanksin-3-acetate(PB3A)on U251 cells and elucidate the underlying molecular mechanisms.Methods Human glioma U251 cells were cultured in vitro.The cells were divided into control group(0μg·mL^-1)and PB3A 2.5,5,10,20,40,60,80μg·mL^-1 groups.The morphological changes of U251 cells were observed by the inverted microscopy.CCK-8 assay was used to detect cell proliferation inhibition rate.EdU was used to detect cell proliferation ability.Flow cytometry was used to detect cell cycle and apoptosis rate.Western Blot was used to detect the expression levels of Bcl-2,Bax,Caspase-3,CyclinB1 and CyclinD1 proteins in U251 cells.Results Compared with the control group,the cell inhibition rates of PB3A in the range of 5-80μg·mL^-1 were significantly increased(P<0.05,P<0.01),and showed a concentration and time dependence.The rates of EdU positive cells of PB3A 10,20,40μg·mL^-1 groups were significantly decreased(P<0.01).Apoptosis rates of PB3A groups were significantly increased(P<0.01).The proportions of G0/G1 phase cells in PB3A groups were significantly decreased(P<0.05,P<0.01),and the proportions of cells in G2/M phase were significantly increased(P<0.05,P<0.01).The protein expression levels of Bax,Caspase-3 were up-regulated significantly in PB3A 20,40μg·mL^-1 groups(P<0.01);while the expression levels of Bcl-2,CyclinB1 were down-regulated significantly(P<0.01).PB3A 20,40μg·mL^-1 groups had no significant effect on the expression level of CyclinD1 protein in U251 cells.Conclusion PB3A can significantly inhibit the proliferation,induce apoptosis and block the cell cycle of U251 cells in G2/M phase,and its effect may be achieved by regulating apoptosis and cycle-related proteins.