叔丁基过氧化氢诱导人正常肝细胞系L02自噬模型的建立及其线粒体应激机制

发布时间：2018-12-15 13:26

【摘要】：目的:建立叔丁基过氧化氢(t-BHP)诱导的人正常肝细胞系L02自噬模型并探讨其中的线粒体应激机制。方法:体外培养L02细胞,用不同浓度(100、200、400、800、1 000μmol/L)t-BHP及线粒体靶向抗氧化剂Mito Q或p38/MAPK特异性抑制剂SB203580进行处理,采用免疫荧光和Western blot检测自噬标志蛋白LC3-II和p62蛋白水平,以及p38/MAPK信号通路的激活情况;Mito-SOX Red染色流式法检测细胞线粒体活性氧(ROS)水平。结果:免疫荧光结果显示t-BHP剂量≥800μmol/L时可明显诱导L02细胞内LC3-II发生聚集。Western blot结果显示,与对照组比较,800和1 000μmol/L t-BHP处理可显著增加LC3-II蛋白水平,并降低p62蛋白水平(P均0.05)。同时线粒体ROS水平在≥400μmol/L t-BHP处理后明显升高,在1 000μmol/L t-BHP处理后p38蛋白磷酸化水平显著增加(P均0.05)。给予线粒体靶向抗氧化剂Mito Q或p38/MAPK特异性抑制剂SB203580预处理后可有效拮抗t-BHP(1 000μmol/L)处理引起的LC3-II和p62蛋白水平改变。结论:我们成功建立了t-BHP诱导体外培养人正常肝细胞系L02的自噬模型,证明线粒体ROS介导了此过程的发生,同时p38/MAPK通路在此过程中发挥了重要作用。
[Abstract]:Aim: to establish an autophagy model of human normal liver cell line L02 induced by tert-Ding Ji hydrogen peroxide (t-BHP) and to explore the mechanism of mitochondrial stress. Methods: L02 cells were cultured in vitro. The cells were treated with different concentrations (100,200,400,000,000U mol/L) t-BHP and mitochondria targeted antioxidant Mito Q or p38/MAPK specific inhibitor SB203580. The levels of LC3-II and p62 proteins and the activation of p38/MAPK signaling pathway were detected by immunofluorescence and Western blot. The levels of reactive oxygen species (Ros) (ROS) in mitochondria were detected by flow cytometry with Mito-SOX Red staining. Results: the results of immunofluorescence showed that the concentration of LC3-II in L02 cells could be induced by the concentration of t-BHP 鈮，

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