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目的研究冬凌草甲素体外诱导胃癌BGC-823细胞凋亡和细胞周期阻滞的作用,阐明其作用机理,为临床应用提供科学依据。方法用MTT方法测定冬凌草甲素体外抑制BGC-823细胞生长的作用。用共聚焦荧光显微镜和流式细胞仪分别观察诱导凋亡的情况。线粒体膜电位和细胞内钙离子浓度分别用荧光探针标记后流式细胞仪测定。凋亡和细胞周期相关蛋白的表达用Western blotting进行测定。结果冬凌草甲素对BGC-823细胞的半数生长抑制浓度IC50值为22.21μmol·L–1,并诱导细胞凋亡,呈现浓度依赖性。此外,能够降低线粒体膜电位,升高细胞内钙离子浓度,激活caspase-3前体。同时,冬凌草甲素能够使得BGC-823细胞阻滞在细胞周期的G2/M期,伴随有cyclinA蛋白的表达下降。在发生凋亡和细胞阻滞之前,观察到p53蛋白的表达增加。结论冬凌草甲素能够诱导BGC-823细胞产生凋亡,并使细胞周期阻滞在G2/M期。其凋亡机理与caspase-3的激活,p53蛋白表达上调及cyclinA表达下调相关。
Objective To study the effect of oridonin on apoptosis and cell cycle arrest of gastric cancer BGC-823 cells in vitro, and to elucidate its mechanism of action, and provide scientific basis for clinical application. Methods MTT assay was used to determine the effect of Rubescensine A on the growth of BGC-823 cells in vitro. Confocal fluorescence microscopy and flow cytometry were used to observe the induction of apoptosis. The mitochondrial membrane potential and intracellular calcium ion concentration were respectively measured with a fluorescent probe and measured by flow cytometry. The expression of apoptosis and cell cycle related proteins was determined by Western blotting. Results Oridonin had a half-maximal growth inhibitory concentration (IC50) of 22.21 μmol·L–1 on BGC-823 cells and induced apoptosis in a concentration-dependent manner. In addition, it can reduce the mitochondrial membrane potential, increase the intracellular calcium ion concentration and activate the caspase-3 precursor. At the same time, Oridonin can make BGC-823 cells arrest in the G2/M phase of the cell cycle, accompanied by a decrease in the expression of cyclinA protein. Increased expression of p53 protein was observed before apoptosis and cell arrest occurred. Conclusion Oridonin can induce apoptosis in BGC-823 cells and arrest cell cycle in G2/M phase. The mechanism of apoptosis was related to the activation of caspase-3, the up-regulation of p53 protein expression and the down-regulation of cyclinA expression.