【摘要】 目的 研究石蒜碱介导人乳腺癌MDA-MB-231细胞自噬及凋亡的作用机制。方法 采用CCK-8法检测石蒜碱对MDA-MB-231细胞增殖的影响；倒置显微镜、荧光显微镜和激光共聚焦扫描显微镜（laser scanning confocal microscopy,LSCM）观察细胞形态；细胞集落实验检测细胞克隆形成能力；划痕实验检测细胞迁移能力；流式细胞仪检测细胞凋亡率、细胞内活性氧（reactive oxygen species,ROS）水平、线粒体膜电位；LSCM检测线粒体膜通透性转换孔（mitochondrial permeability transition pore,MPTP）开放情况；Western blotting检测线粒体自噬相关蛋白[线粒体内膜转位酶（translocase of inner membrane,TIM）、线粒体外膜转位酶（translocase of outer membrane,TOM）、E3泛素连接酶（E3 ubiquitin protein ligase,PARKIN）、PTEN诱导的激酶1(PTEN induced putative kinase 1,PINK1)、微管相关蛋白轻链3(microtubule-associated protein light chain 3,LC3-B)、p62]和凋亡相关蛋白[半胱氨酸天冬氨酸蛋白酶-3(cystein-asparate protease-3,Caspase-3)、Bcl-2相关X蛋白（Bcl-2 associated X protein,Bax）、B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)、细胞色素C(cytochrome-C,CytC)]的表达。结果 石蒜碱对MDA-MB-231细胞具有生长抑制作用，其半数抑制浓度（half inhibitory concentration,IC50）值为6.21μmol/L；石蒜碱能够改变细胞形态，部分细胞出现凋亡迹象；石蒜碱能够显著抑制细胞的增殖和迁移能力（P<0.01）；与对照组比较，随着石蒜碱给药浓度的增加，细胞凋亡率随之升高（P<0.01）,ROS水平升高（P<0.01），线粒体膜电位下降（P<0.01）,MPTP开放（P<0.01）；石蒜碱给药48 h后，细胞内TIM、TOM、p62和Bcl-2蛋白表达量下调（P<0.01）,PARKIN、PINK1、LC3-B、Caspase-3、Bax和Cyt-C蛋白表达量上调（P<0.01），且呈浓度相关性。结论 石蒜碱对MDA-MB-231细胞具有生长抑制作用，并可通过调控线粒体氧化损伤介导MDA-MB-231细胞的自噬及凋亡。更多还原

【Abstract】 Objective To investigate the mechanism of lycorine on autophagy and apoptosis in human breast cancer MDA-MB-231cell. Methods The effect of lycorine on MDA-MB-231 cell proliferation was detected by CCK-8; Cell morphology was observed by inversion microscopy, fluorescence microscopy and laser scanning confocal microscopy(LSCM); The cell clone formation ability was detected by cell colony assay; The cell migration ability was examined by scratch assay; The apoptosis rate, intracellular reactive oxygen species(ROS) level and mitochondrial membrane potential were determined by flow cytometry; The opening of mitochondrial membrane permeability transition pore(MPTP) was detected by LSCM; The expressions of mitochondrial autophagy-related proteins [translocase of inner membrane(TIM), translocase of outer membrane(TOM), E3 ubiquitin protein ligase(PARKIN), PTEN induced putative kinase 1(PINK1), microtubule-associated protein light chain 3(LC3-B), p62], and apoptosis-related proteins [cystein-asparate protease-3(Caspase-3), Bcl-2 associated X protein(Bax), B-cell lymphoma-2(Bcl-2), cytochrome-C(Cyt-C)] were detected by Western blotting. Results Lycorine had a proliferative inhibitory effect on MDA-MB-231 cells, with a half inhibitory concentration(IC50) value of 6.21 μmol/L; Lycorine could change cell morphology, and some cells showed signs of apoptosis; Lycorine could significantly inhibit the proliferation and migration capacity of MDA-MB-231 cells(P < 0.01); Compared with control group, with the increase of lycorine administration concentration, cell apoptosis rate was increased(P < 0.01), ROS level was increased(P < 0.01),mitochondrial membrane potential was decreased(P < 0.01), and MPTP was open(P < 0.01). After 48 h treatment of lycorine, the expressions of TIM, TOM, p62 and Bcl-2 protein were down-regulated(P < 0.01), and the expressions of PARKIN, PINK1, LC3-B,Caspase-3, Bax and Cyt-C were up-regulated(P < 0.01) in a concentration-dependent manner. Conclusion Lycorine has growth inhibition effect on MDA-MB-231cells, and induces autophagy and apoptosis of MDA-MB-231 cells through the regulation of mitochondrial oxidative damage.更多还原