华蟾素对MDA-MB-231细胞内钙离子和细胞膜电位的影响

摘要

目的:研究华蟾素对乳腺癌MDA-MB-231细胞内钙离子和细胞膜电位的影响及其作用机制.方法:根据原子力显微镜 (AFM) 高分辨率和高灵敏度的特点,从形态学上研究华蟾素对MDA-MB-231细胞的凋亡作用; 分别用DiBAC4 (3) 及Fluo-3/AM对MDA-MB-231细胞进行细胞膜电位和钙离子的荧光染色,然后用流式细胞仪检测不同浓度 (0、6.25、12.5、25、37.5、50 μg·mL-1) 华蟾素对细胞膜电位和细胞内钙离子的浓度变化.结果:原子力显微镜观察到华蟾素作用后,MDA-MB-231细胞形态及超微结构发生了明显变化,细胞表面粗糙度增加,从形态学上研究华蟾素的促凋亡作用.同时,用流式细胞仪检测到华蟾素作用细胞后,细胞膜电位发生去极化,并且细胞内钙离子浓度增加.结论:华蟾素作用于MDA-MB-231细胞,使得细胞发生去极化,从而激活了电压依赖型钙离子通道的开放,Ca2+是细胞内信号传导过程中重要的第二信使,进而介导了MDA-MB-231细胞的凋亡.%Objective: To explore the effect of cinobufacini on the breast cancer cell line MDA-MB-231. The effects of cinobufa-cini on intracellular calcium ions and cell membrane potential were studied. Methods: Atomic force microscopy-based technology was used to detect the changes in the morphologic properties of cells at the nanometer scale. The membrane potential of breast cancer cells was determined by staining with fluorescence probe DiBAC4(3) and the calcium ions were stained with fluorescence probe Fluo-3/AM in each group, and then FCM technology was utilized to measure the changes in fluorescence intensity. Results: The AFM morphologic data showed that the MDA-MB-231 cells in the control group were swollen, but after cinobufacini treatment, the size of cells decreased and the nucleoli became crimpled. Flow cytometry data indicated the cell membrane potential and the calcium ion concentration increased significantly after cinobufacini treatment with different concentrations. Conclusion: Cinobufacini induces the apoptosis of MDA-MB-231 cells, causes the cell membrane potential to depolarize; then, calcium ion channels are opened and calcium ions influx into the cells. Finally, the calcium ion acted as secondary messengers for cell-mediated apoptosis.