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(80.0 μmol·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)、Ca2+离子水平。采用胞外Ca2+螯合剂胞(1 mmol·L-1 EGTA)和胞内Ca2+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、Ca2+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Ca2+和Cyt C释放有关。
Abstract
OBJECTIVE To investigate the mitochondrial damage and the possible mechanism in the process of MCF-7 cell apoptosis which induced by quercetin. METHODS The cell apoptosis model was established with MCF-7 cells induced by quercetin. The morphological characteristics of apoptotic MCF-7 cells were observed by transmission electron microscope. Fluorescent probes DCFH-DA and Fluo-8-AM were used to detect the invaded cells′ reactive oxygen species (ROS) and intracellular Ca2+ concentration, respectively. The calcium channel blockers of extracellular and intracellular were used to inhibit apoptosis induced by quercetin in MCF-7 cells, the cellular apoptosis rates of which were measured by flow cytometry. Western blot assay was used to detect the expressions of Cyt C. RESULTS Quercetin can induce MCF-7 cell mitochondria apoptosis. The spherical swelling, mitochondrial crest disappear and the formation of cavity were happened to those cells. Intracellular ROS and Ca2+ increased. The apoptosis induced by quercetin can be inhibit by intracellular calcium blockers ryanodine. After incubation with ryanodine, apoptosis rates fell by 55.4% at 24 h, 55.4% at 48 h and 39.9% at 72 h, respectively. The cell apoptosis induced by quercetin was not inhibited by EDTA. The expression of Cyt C was increased in the cytoplasm but not mitochondria after cultured the MCF-7 cell with EDTA.CONCLUSION The mitochondrial damage plays an important role in the MCF-7 apoptosis which induced by quercetin, and its possible mechanism may related to the elevated levels of ROS and the release of Ca2+ and Cyt C in the cytoplasm. Meanwhile, Cyt C in the cytosol is potential critical signaling molecular in the process of apoptosis.
关键词
槲皮素 /
MCF-7细胞 /
细胞凋亡 /
线粒体 /
活性氧 /
细胞色素C
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Key words
quercetin /
MCF-7 cell lines /
apoptosis /
mitochondria /
reactive oxygen species /
Cyt C
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中图分类号：
R378.3
R965
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参考文献
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脚注
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基金
国家级大学生创新创业训练计划资助项目(201610354023);2012年度嘉兴市重点科技创新团队资助项目(2012-03)
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