摘要
目的:探究紫外线抵抗相关基因(UVRAG)对金黄色葡萄球菌(SA)诱导HeLa细胞自噬及胞内菌存活的影响。方法:通过SA刺激HeLa细胞建立体外感染模型,分别用UVRAG小干扰RNA(siRNA)及pCI-neo-HA-hUVRAG质粒转染HeLa细胞。Western blot法检测自噬相关蛋白LC3B、p62及UVRAG的表达水平。用EGFP-LC3质粒及pCI-neo-HA-hUVRAG质粒共转染HeLa细胞后SA感染细胞,激光共聚焦显微镜观察EGFP-LC3的点状聚集。分别用UVRAG siRNA及pCI-neo-HA-hUVRAG质粒转染HeLa细胞后SA感染细胞,平板计数法计算胞内SA数量。结果:SA感染HeLa细胞能够引起自噬水平升高以及UVRAG的表达升高,UVRAG siRNA能够抑制LC3Ⅱ的表达,促进p62的表达。过表达UVRAG能够促进LC3Ⅱ的表达,抑制p62的表达。过表达UVRAG使EGFP-LC3的点状聚集增多,并且抑制胞内菌的存活,UVRAG siRNA促进胞内菌的复制。结论:SA感染宿主细胞诱导UVRAG表达的增加可以增强自噬水平并且抑制胞内菌的存活。
Objective:In order to investigate the effect of UV radiation resistance-associated gene(UVRAG)on autophagy and intracellular survival of HeLa cells induced by staphylococcus aureus(SA).Methods:HeLa cells were stimulated by SA to establish an in vitro infection model.HeLa cells were transfected with the UVRAG siRNA and the pCI-neo-HA-hUVRAG respectively.LC3B,p62 and UVRAG were detected by Western blot.Infected with SA after co-transfection of HeLa cells with EGFP-LC3 plasmid and pCI-neo-HA-hUVRAG plasmid,the number of EGFP-LC3 puncta was observed by laser confocal microscope.HeLa cells were infected with SA after transfection with UVRAG siRNA and pCI-neo-HA-hUVRAG plasmid,respectively.The number of intracellular bacteria was determined by colony formation assay(CFU)counting.Results:SA infection of HeLa cells can cause increased autophagy and UVRAG expression.Suppression of UVRAG inhibited LC3Ⅱand enhanced p62 protein level.Over-expression of UVRAG enhanced LC3Ⅱand inhibited p62 protein level.Over-expression of UVRAG enhanced the number of EGFP-LC3 puncta and inhibited the number of intracellular bacteria.Suppression of UVRAG enhanced the number of intracellular bacteria replication.Conclusion:Increased UVRAG expression induced by SA infection of host cells can enhance autophagy level and inhibit intracellular bacterial survival.
作者
杨彬
王郅杰
么宏强
邓天健
额尔敦木图(指导)
YANG Bin;WANG Zhi-Jie;YAO Hong-Qiang;DENG Tian-Jian;Erdunmtu(College of Veterinary Medicine,Inner Mongolia Agricultural University,Huhhot 010018,China)
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2020年第8期914-918,共5页
Chinese Journal of Immunology
基金
黑龙江省青年科学基金(QC2016030)
地区科学基金(31860693)资助。
关键词
金黄色葡萄球菌
紫外线抵抗相关基因
自噬
Staphylococcus aureus
Radiation resistance-associated gene
Autophagy