[关键词]
[摘要]
本文研究了棒曲霉素抑制人胚肾细胞增殖与诱导其凋亡的作用。不同浓度的棒曲霉素作用于人胚肾细胞293后,采用乳酸脱氢酶释放检测棒曲霉素对细胞增殖的抑制作用。通过扫描电镜和荧光显微镜观察了细胞形态学的变化,Annexin V-EGFP/PI标记检测细胞凋亡率,JC-1线粒体膜电位荧光探针检测了细胞线粒体膜电位的变化。实时荧光定量PCR测定了线粒体相关基因FIS1、ASL、SLC25A6、COX17的表达水平。结果表明,棒曲霉素可显著抑制人胚肾细胞293增殖并诱导凋亡,具有明显的量效关系,2.5、5、7.5、10和15 μM棒曲霉素对人胚肾细胞293的抑制率分别为8.1%,18.2%,31.0%,42.2%和63.1%;5 μM棒曲霉素处理0,3,10,24 h,细胞的存活率分别为94.2%、78.1%、47.4%。5 μM和10 μM的棒曲霉素处理8h后,细胞凋亡率分别为16.4%,和20.1%。线粒体膜电位和线粒体相关基因的表达水平的变化表明棒曲霉素可能通过线粒体途径引起人胚肾细胞293凋亡。
[Key word]
[Abstract]
To study the mechanism of patulin on the proliferation and apoptosis of human embryonic kidney cells, HEK293 cells were cultured and treated with different concentrations of patulin in vitro. The effect of patulin on cell proliferation level was examined by LDH cytotoxicity assay. The morphological changes in patulin-treated HEK293 cells were observed using scanning electron microscopy and fluorescence microscopy. The cell apoptosis rate was detected by Annexin V-EGFP/PI double staining. The mitochondrial membrane potential was measured by a JC-1 probe. The expression levels of the mitochondrial related genes FIS1, ASL, SLC25A6, and COX17 were measured by real-time fluorescent quantitative PCR. The results showed that PAT inhibited the growth of HEK293 cells in a dose-dependent manner and resulted in apoptosis in HEK293 cells. Treatments with 2.5, 5, 7.5, 10, and 15 μM PAT for 8 h resulted in significant decreases in cell viability by 8.1%, 18.2%, 31.0%, 42.2%, and 63.1%, respectively. When treated with 5 μM patulin for 0, 3, 10, and 24 h, the cell survival rates were 94.2%, 78.1%, and 47.4%, respectively. After treatment with 5 and 10 μM PAT for 8 h, the number of apoptotic cells increased in a dose-dependent manner by 16.4% and 20.1%. The changes in the expression levels of mitochondrial membrane potential and mitochondrial genes indicate that patulin may cause HEK293 apoptosis through mitochondrial pathways.
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[基金项目]
国家自然基金(31201286);中央高校基本科研创新一般项目(2014YB021)