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[摘要]
目的 研究石蒜碱诱导人肝癌HepG-2细胞凋亡的作用及其机制。方法 噻唑蓝(MTT)法检测石蒜碱对HepG-2细胞的生长抑制作用;倒置显微镜、荧光显微镜观察HepG-2细胞凋亡形态;透射电镜观察HepG-2细胞超微结构;流式细胞术检测HepG-2细胞凋亡率;激光共聚焦扫描显微镜检测线粒体膜电位;激光共聚焦显微镜检测HepG-2细胞线粒体膜通透性转换孔(MPTP);酶标仪检测Caspase-3蛋白活性;Western blotting法检测细胞内凋亡相关蛋白Bcl-2、Bax、Cyt-C、Caspase-9的表达。结果 石蒜碱对HepG-2细胞具有生长抑制作用,其半数抑制浓度(IC50)为5.73 μmol/L;形态学观察发现石蒜碱作用48 h后,细胞生长密度变疏、皱缩,且细胞生长呈现不规则形状,出现凋亡小体,随着给药浓度升高,凋亡小体也逐渐增多;透射电镜下可见细胞出现典型的凋亡特征;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,线粒体膜电位下降,MPTP开放;石蒜碱作用48 h后,HepG-2细胞内Caspase-3蛋白相对活性升高,细胞内Cyt-C蛋白、Caspase-9蛋白表达水平升高,Bcl-2蛋白表达量降低,Bax蛋白表达量升高,且呈浓度依赖性关系,与对照组比较,具有统计学意义。结论 石蒜碱对HepG-2细胞具有生长抑制作用,并可通过启动线粒体途径诱导HepG-2细胞发生凋亡。
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[Abstract]
Objective To investigate the effect and mechanism of lycorine apoptosis in the human Liver cancer cell line HepG-2. Methods The inhibitory effect of Lycorine on HepG-2 cell growth was evaluated by the dimethylthiazol tetrazolium assay. Morphological apoptotic changes were characterized using an inverted microscope, Hoechst 33258 fluorescence staining and cell ultrastructure. The rate of cell apoptosis was determined by flow cytometry. Mitochondrial membrane potential and intracellular fluorescent intensity of MMP were detected using laser scanning confocal fluorescence microscopy. Expression of the proteins Bcl-2, Bax, cytochrome C, and Caspase-9 was assessed by western blotting. The activity of Caspase-3 protein was quantified using a Caspase-3 activity kit. Results Lycorine inhibited the growth of HepG-2 cell line with an IC50 of 5.73 μmol/L. After 48 h lycoris radiata alkali treatment, morphological observation indicated that, the density of growth cell became thin, cell shrinkage and cell growth form an irregular shape, tend to appear apoptotic body. Moreover, with the drug concentration increases, the apoptotic body also gradually increased; Typical apoptosis characteristics of visible cells were observed under transmission electron microscopy. Compared with control group, with the increase of concentration of lycoris radiata alkali dosage, cell apoptosis rate increased, mitochondrial membrane potential decreased and MPTP membrane was open. After 48 h treatment of lycorine, relative activity of Caspase-3 increased in the HepG-2 cells, the expression of cytochrome C, Caspase-9 and Bax on the protein level were upregulated, and the expression of Bcl-2 protein was downregulated, and respectively into concentration dependence relationship, with statistical significance. Conclusion Lycorine radiata had growth inhibition effect on human liver HepG-2 cells, and induced apoptosis of HepG-2 cells through mitochondrial pathway.
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[基金项目]
国家自然科学基金资助项目(81274067);哈尔滨市科技局后备人才项目(2016RQXJ124);哈尔滨市科技局优秀学科带头人项目(2016RAXXJ064);黑龙江省高校创新人才项目(UNPYSCT-2017208);哈尔滨商业大学校级科研项目(17XN008)