反油酸对人神经母细胞瘤细胞系SH-SY5Y细胞线粒体功能和细胞自噬的影响
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(首都医科大学公共卫生学院,北京 100069)

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范荣 女 硕士生 研究方向为营养与老年相关慢性疾病 E-mail:fanrong53782@sina.com通信作者:┣┣(中)通信作者┫┫麻微微 女 副教授 研究方向为营养与老年相关慢性疾病 E-mail:weiweima@ccmu.edu.cn

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国家自然科学基金项目(81472982,81773406);北京市教育委员会科技发展计划一般项目(KM201710025007)


Effect of elaidic acids on mitochondrial function and autophagy in SH-SY5Y cells
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(School of Public Health,Capital Medical University,Beijing 100069,China)

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    摘要:

    目的 研究反油酸对人神经母细胞瘤细胞系SH-SY5Y细胞线粒体功能和细胞自噬的影响。方法 体外培养人神经母细胞瘤细胞系SH-SY5Y细胞,加入不同剂量(0、10、20、50和100 μmol/L)的反油酸,24 h后收集细胞,采用透射电镜观察细胞自噬小体;采用细胞计数试剂盒8(CCK-8)检测细胞活力;采用实时荧光定量聚合酶链式反应(PCR)和蛋白免疫印迹(western blot)方法检测细胞自噬相关的基因和蛋白表达水平。结果 透射电镜观察细胞结构发现,50和100 μmol/L反油酸可使细胞内出现自噬小体,并可以引起细胞活力下降,差异均有统计学意义(P<0.01);100 μmol/L反油酸作用24和48 h后,细胞活力明显降低,差异均有统计学意义(P<0.05);100 μmol/L反油酸可使SH-SY5Y细胞中细胞色素C(cytochrome c,cyt-c)基因mRNA表达水平明显降低,差异有统计学意义(P<0.05);随着反油酸浓度的升高,细胞浆中cyt-c蛋白表达水平逐渐升高,差异均有统计学意义(P<0.05)。但是不同剂量的反油酸对SH-SY5Y细胞自噬微管相关蛋白轻链3B(LC3B)基因、Beclin1基因、p62基因、自噬相关基因5(atg5)、自噬相关基因12(atg12)mRNA水平表达差异无统计学意义(P>0.05);但与对照组比较,50和100 μmol/L反油酸可使SH-SY5Y细胞LC3B和Beclin1蛋白表达水平逐渐上调,差异均有统计学意义(P<0.05)。结论 反油酸可以引起人神经母细胞瘤细胞系SH-SY5Y细胞活力下降,可能与其引起细胞自噬小体形成,上调自噬相关蛋白表达水平,促进线粒体cyt-c蛋白释放入细胞浆有关。

    Abstract:

    Objective The aim of this study was to determine the effects of elaidic acid on mitochondrial function and autophagy in the human neuroblastoma cell line SH-SY5Y. Methods SH-SY5Y cells were cultured in vitro and treated with different concentrations (0,0, 20,0 and 100 μmol/L) of elaidic acid for 24 hours. The morphology of cells and intracellular autophagosomes were observed by transmission electron microscope. The cell viability was detected by using CCK-8 detection kit. Real-time polymerase chain reaction(PCR)and western blot were used to measure the autophagy-related genes and proteins expression. Results Observation of cell structures found that intracellular autophagosomes formed with 50 and 100 μmol/L of elaidic acid. 50 and 100 μmol/L of elaidic acid might result in a decline in cell viability (P<0.01). 100 μmol/L of elaidic acid significantly decreased cell viability both after 24 and 48 h (P<0.05). 100 μmol/L of elaidic acid significantly decreased the cytochrome c (cyt-c) gene mRNA expression in SH-SY5Y cells (P<0.05). The expression level of cyt-c protein in the cytoplasm was gradually elevated with the increase of elaidic acid concentrations (P<0.05). But the differences with regard to the LC3B, Beclin1, p62, atg5 and atg12 genes mRNA expression levels of SH-SY5Y cells were not significant across different doses(P>0.05). 50 and 100 μmol/L of elaidic acid gave rise to the expression of LC3B and Beclin1 proteins up-regulated compared with the normal group (P<0.05). Conclusion Elaidic acid contributes to the decrease of SH-SY5Y cells viability, which may be associated with the formation of autophagosomes, up-regulating autophagy protein expression, promoting the changes of mitochondrial permeability and releasing mitochondrial cyt-c protein into cytoplasm.

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范荣,孙昊,席元第,周催,吕晨艳,杨春,蔡夏夏,肖荣,麻微微.反油酸对人神经母细胞瘤细胞系SH-SY5Y细胞线粒体功能和细胞自噬的影响[J].中国食品卫生杂志,2018,30(3):223-228.

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  • 收稿日期:2018-04-02
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  • 在线发布日期: 2018-06-20
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