發(fā)布時間：2018-12-15 13:26

【摘要】：目的:建立叔丁基過氧化氫(t-BHP)誘導(dǎo)的人正常肝細(xì)胞系L02自噬模型并探討其中的線粒體應(yīng)激機(jī)制。方法:體外培養(yǎng)L02細(xì)胞,用不同濃度(100、200、400、800、1 000μmol/L)t-BHP及線粒體靶向抗氧化劑Mito Q或p38/MAPK特異性抑制劑SB203580進(jìn)行處理,采用免疫熒光和Western blot檢測自噬標(biāo)志蛋白LC3-II和p62蛋白水平,以及p38/MAPK信號通路的激活情況;Mito-SOX Red染色流式法檢測細(xì)胞線粒體活性氧(ROS)水平。結(jié)果:免疫熒光結(jié)果顯示t-BHP劑量≥800μmol/L時可明顯誘導(dǎo)L02細(xì)胞內(nèi)LC3-II發(fā)生聚集。Western blot結(jié)果顯示,與對照組比較,800和1 000μmol/L t-BHP處理可顯著增加LC3-II蛋白水平,并降低p62蛋白水平(P均0.05)。同時線粒體ROS水平在≥400μmol/L t-BHP處理后明顯升高,在1 000μmol/L t-BHP處理后p38蛋白磷酸化水平顯著增加(P均0.05)。給予線粒體靶向抗氧化劑Mito Q或p38/MAPK特異性抑制劑SB203580預(yù)處理后可有效拮抗t-BHP(1 000μmol/L)處理引起的LC3-II和p62蛋白水平改變。結(jié)論:我們成功建立了t-BHP誘導(dǎo)體外培養(yǎng)人正常肝細(xì)胞系L02的自噬模型,證明線粒體ROS介導(dǎo)了此過程的發(fā)生,同時p38/MAPK通路在此過程中發(fā)揮了重要作用。
[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，

本文編號：2380693