染料木素衍生物对U87-MG细胞凋亡机制的研究

Study on mechanism of genistein derivatives apoptosis in U87-MG cells

目的:研究染料木素衍生物(Ged)对U87-MG细胞的凋亡机制。方法:实验分为对照组,染料木素衍生物组(10、20、40μmol·L^(-1));以MTT法检测细胞体外抗增殖活性,流式细胞仪检测细胞的凋亡状况,荧光定量PCR仪检测细胞内Bax mRNA、Bcl-2 mRNA、Caspase-3 mRNA水平,激光共聚焦检测线粒体膜电位变化和活性氧含量,酶标仪检测Caspase-3酶的活性。结果:随染料木素衍生物浓度的增加,对U87-MG细胞的抑制性增强,细胞的凋亡量成浓度依赖性,IC50为20.23μmol·L^(-1);荧光定量PCR结果显示,Bax mRNA、Bcl-2 mRNA、Cyt c、Caspase-3 mRNA的基因表达水平显著增加(P<0.05);当浓度为20μmol·L^(-1)时,线粒体膜电位降低显著;随着细胞的凋亡,活性氧的生成也与药物浓度呈依赖性;Caspase-3酶的活性在药物浓度为40μmol·L^(-1)时,达到极显著水平(P<0.01)。结论:染料木素衍生物(Ged)能抑制U87-MG细胞的增殖,增强活性氧的簇产生、线粒体膜电位下降,随细胞凋亡量的增加,Caspase-3酶的活性增强,染料木素衍生物具有较强的抗癌活性。

Objective:To study the mechanism of genistein derivatives apoptosis in U87-MG cells.Methods:The experiments were divided into a control group and genistein derivative groups(10,20,40μmol·L^(-1)).In vitro anti-proliferative activity of cells was evaluated by MTT.Apoptosis of cells was characterized by flow cytometry.Quantitative fluorescent PCR instrument was used to detect the levels of Bax mRNA,Bcl-2 mRNA,Cyt c and Caspase-3 mRNA in the cells.Laser confocal was applied to detect the mitochondrial membrane potential changes.Reactive oxygen content,microplate reader are used to detect the activity of Caspase-3 enzyme.Results:As the concentration of genistein derivatives increases,the inhibitory effect on U87-MG cells increases.And the amount of cell apoptosis is concentration-dependent,with an IC50 of 20.23μmol·L^(-1).Quantitative fluorescence PCR results showed that the gene expression level of Bax mRNA,Bcl-2 mRNA and Caspase-3 mRNA increased significantly(P<0.05).When the concentration is 20μmol·L^(-1),the mitochondrial membrane potential decreases significantly.The generation of reactive oxygen species was also dependent on the concentration of the drug with the apoptosis of cells.The activity of the Caspase-3 enzyme reached the extreme Significant level(P<0.01)when the drug concentration was 40μmol·L^(-1).Conclusion:The genistein derivative(Ged)can inhibit the proliferation of U87-MG cells,enhance the generation of reactive oxygen clusters,and reduce the mitochondrial membrane potential.With the increase of the amount of apoptosis,the activity of Caspase-3 enzyme increases.The genistein derivatives have strong anticancer activities.