金黄色葡萄球菌对巨噬细胞自噬相关蛋白表达的影响
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摘要
巨噬细胞是机体重要的免疫防御细胞,本试验探讨金黄色葡萄球菌感染对巨噬细胞自噬相关蛋白表达的影响,为进一步研究自噬在金黄色葡萄球菌侵袭过程中的作用提供理论依据。采用金黄色葡萄球菌侵袭RAW264.7细胞,运用Western blot分别于侵袭0,15,30,45,60,90,120 min检测细胞内自噬相关蛋白Beclin1、Atg5、LC3、p62的表达水平,激光共聚焦技术检测细胞内LC3聚点,并用透射电镜观察细胞超微结构。结果显示,与侵袭0 min相比,Beclin1、Atg5、LC3、p62表达水平在各时间点均升高(P<0.01),且呈一定的时间-效应关系。侵袭45 min时,细胞内LC3蛋白聚点增多,且单个细胞的荧光强度增强。透射电镜观察显示金黄色葡萄球菌侵入细胞内,并形成明显的吞噬泡。故金黄色葡萄球菌侵袭巨噬细胞,促进了细胞自噬的发生和发展,自噬参与了金黄色葡萄球菌的侵袭过程。
Macrophage is an important immune-defense cells in the body.The research aimed to explore the effects of Staphylococcus aureus on autophagy-related proteins expression in macrophages,which could provide the theoretical foundation about the autophagy function during the S.aureus invasion.RAW264.7 cells was inoculated with S.aureus. Autophagy-related proteins expression of Beclin1,ATG5,LC3 and p62 was detected with Western blot at 0,15,30,45,60,90 and 120 min respectively.LC3 accumulation points were observed with laser scanning confocal microscope.In the meanwhile,cell ultrastructure was observed by TEM.Results showed that expression levels of proteins including Beclin1,Atg5,LC3,and p62 increased at different time points compared with 0 min(P<0.01).LC3 accumulation points in the cells increased and the fluorescence intensity of individual cells also increased.Many S.aureus were caught in cells and phagocytic vesicles were also formed with TEM.So,autophagy of macrophages were accelerated with S.aureus invasion and autophagy was also involved in the invasion of S.aureus.
Macrophage is an important immune-defense cells in the body.The research aimed to explore the effects of Staphylococcus aureus on autophagy-related proteins expression in macrophages,which could provide the theoretical foundation about the autophagy function during the S.aureus invasion.RAW264.7 cells was inoculated with S.aureus. Autophagy-related proteins expression of Beclin1,ATG5,LC3 and p62 was detected with Western blot at 0,15,30,45,60,90 and 120 min respectively.LC3 accumulation points were observed with laser scanning confocal microscope.In the meanwhile,cell ultrastructure was observed by TEM.Results showed that expression levels of proteins including Beclin1,Atg5,LC3,and p62 increased at different time points compared with 0 min(P<0.01).LC3 accumulation points in the cells increased and the fluorescence intensity of individual cells also increased.Many S.aureus were caught in cells and phagocytic vesicles were also formed with TEM.So,autophagy of macrophages were accelerated with S.aureus invasion and autophagy was also involved in the invasion of S.aureus.
引文
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[3] MESTRE M B,COLOMBO M I.Staphylococcus aureus promotes autophagy by decreasing intracellular cAMP levels[J].Autophagy,2012,8(12):1865-1867.
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[6] LIANG X H,JACKSON S,SEAMAN M,et al.Induction of autophagy and inhibition of tumorigenesis by beclin 1[J].Nature,1999,402(6762):672-676.
[7] 安雅男,施晓晨,王超,等.黄曲霉毒素B1诱导MEK/ERK/ROS介导的巨噬细胞自噬反应[J].吉林农业大学学报,2017,39(1):106-112.
[8] 苏晶晶,李姿,兰云刚,等.自噬相关蛋白Beclin-1对PHEV复制增殖的影响[J].中国兽医学报,2018,38(5):956-961.
[9] MCMANUS S,ROUX S.The adaptor protein p62/SQSTM1 in osteoclast signaling pathways[J].J Mol Signal,2012,7(1):1.
[10] JOHANSEN T,LAMARK T.Selective autophagy mediated by autophagic adapter proteins[J].Autophagy,2011,7(3):279-296.
[11] PARK S,CHOI S G,YOO S M,et al.Choline dehydrogenase interacts with SQSTM1/p62 to recruit LC3 and stimulate mitophagy[J].Autophagy,2014,10(11):1906-1920.
[12] KOMATSU M,KUROKAWA H,WAGURI S,et al.The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf2 through inactivation of Keap1[J].Nat Cell Biol,2010,12(3):213-223.
[13] KANG S A,PACOLD M E,CERVANTES C L,et al.mTORC1 phosphorylation sites encode their sensitivity to starvation and rapamycin[J].Science,2013,341(6144):1236566.
[14] NEUMANN Y,BRUNS S A,ROHDE M,et al.Intracellular Staphylococcus aureus eludes selective autophagy by activating a host cell kinase[J].Autophagy,2016,12(11):2069-2084.
[15] NGU M,HIRATA E,SUZUKI K.Visualization of Atg3 during autophagosome formation in Saccharomyces cerevisiae[J].J Biol Chem,2015,290(13):8146-8153.
[16] 孙雅婧,郭青龙.细胞自噬的研究进展[J].药学与临床研究,2012,20(3):236-239.
[17] PARK S,CHOI J,BIERING S B,et al.Targeting by autophagy proteins (TAG):targeting of IFNG-inducible GTPases to membranes by the LC3 conjugation system of autophagy[J].Autophagy,2016,12(7):1153-1167.
[18] STREETER A,MENZIES F M,RUBINSZTEIN D C.LC3-II Tagging and Western blotting for monitoring autophagic activity in mammalian cells[J].Methods Mol Biol,2016,1303:161-170.