论文部分内容阅读
目的探讨槲皮素(quercetin)诱导人乳腺癌MCF-7细胞凋亡过程中,线粒体损伤作用及其可能机制。方法建立槲皮素(80.0μmol·L~(-1))诱导MCF-7细胞凋亡模型。采用透射电镜观察槲皮素诱导MCF-7细胞凋亡过程中线粒体病变情况。荧光探针DCFH-DA、Fluo-8/AM检测槲皮素处理的MCF-7细胞内活性氧(ROS)、Ca~(2+)离子水平。采用胞外Ca~(2+)螯合剂胞(1mmol·L~(-1)EGTA)和胞内Ca~(2+)阻断剂(1 nmol·L~(-1)ryanodine)进行阻断试验,流式细胞术检测阻断前、后细胞凋亡情况。采用Western blot检测细胞浆、线粒体细胞色素C(Cyt C)的表达。结果槲皮素可诱导MCF-7细胞线粒体损伤,呈现球形肿胀、线粒体嵴消失、大量空泡形成。细胞内ROS、Ca~(2+)水平升高。胞内钙离子阻断剂ryanodine可抑制槲皮素的细胞凋亡作用,细胞凋亡率分别下降55.4%(24 h),30.0%(48 h)和39.9%(72 h),胞外钙离子螯合剂EGTA则无抑制作用。处理24,48和72 h的MCF-7细胞,胞浆内Cyt C表达升高,线粒体Cyt C表达变化不大。结论线粒体损伤在槲皮素诱导MCF-7细胞凋亡中起重要作用,其机制可能与胞内ROS的升高,胞浆内Ca~(2+)和Cyt C释放有关。
Objective To investigate the mitochondrial damage induced by quercetin in human breast cancer cell line MCF-7 and its possible mechanism. Methods The apoptosis model of MCF-7 cells induced by quercetin (80.0 μmol·L -1) was established. Transmission electron microscopy was used to observe the mitochondrial pathological changes induced by quercetin in MCF-7 cells. Fluorescence probe DCFH-DA and Fluo-8 / AM were used to detect the levels of reactive oxygen species (ROS) and Ca 2+ in quercetin-treated MCF-7 cells. The extracellular Ca 2+ chelator cell (1 mmol·L -1 EGTA) and intracellular Ca 2+ blockers (1 nmol·L -1 ryanodine) were used to block Test, flow cytometry before and after blocking the apoptosis of cells. Western blot was used to detect the expression of cytoplasmic and mitochondrial cytochrome C (Cyt C). Results Quercetin induced mitochondrial damage in MCF-7 cells, with swelling of the sphere, disappearance of the mitochondrial cristae and formation of a large number of vacuoles. Intracellular ROS, Ca ~ (2+) levels increased. The intracellular calcium ion blocker ryanodine can inhibit the apoptosis of quercetin, the apoptotic rates decreased by 55.4% (24 h), 30.0% (48 h) and 39.9% (72 h), respectively. The extracellular calcium Chelator EGTA has no inhibitory effect. At 24, 48 and 72 h, the expression of Cyt C in cytoplasm increased, and the expression of Cyt C in mitochondria changed little. Conclusion Mitochondrial injury plays an important role in the apoptosis of MCF-7 cells induced by quercetin. The mechanism may be related to the increase of intracellular ROS and the release of cytosolic Ca 2+ and Cyt C.