创伤弧菌溶细胞素膜成孔肽对巨噬细胞溶酶体和ROS水平及细胞活性的影响  

Effects of Vibrio vulnificus cytolysin membrane pore-forming peptide on lysosome formation and ROS production in macrophages and its cytotoxicity

作　　者：郑易[1] 黄显辉[1] 谢旦立[1] 楼永良[1] Zheng Yi;Huang Xianhui;Xie Danli;Lou Yongliang(School of Laboratory Medicine and Life Science,Wenzhou Medical University,Wenzhou 325035,China)

机构地区：[1]温州医科大学检验医学院,生命科学学院,325035

出　　处：《中华微生物学和免疫学杂志》2020年第11期852-856,共5页Chinese Journal of Microbiology and Immunology

摘　　要：目的研究重组创伤弧菌溶细胞素膜成孔多肽(rMpf)对小鼠J774A.1巨噬细胞株的细胞毒性作用及对活性氧簇(ROS)和溶酶体的影响,并了解其作用巨噬细胞后的胞内定位情况。方法利用大肠埃希菌表达系统表达rMpf,体外作用于小鼠J774A.1巨噬细胞株。流式细胞术分析rMpf对小鼠J774A.1巨噬细胞株的细胞毒性作用及对ROS和溶酶体的影响。激光共聚焦显微镜观察其胞内定位情况。结果低浓度(4μg/ml)rMpf作用小鼠J774A.1巨噬细胞后,其存活率、胞内ROS和溶酶体无明显变化。20和60μg/ml rMpf作用J774A.1巨噬细胞后,细胞存活率、ROS和溶酶体均明显下降。此外,rMpf能进入J774A.1巨噬细胞并定位于细胞质和细胞核,且呈剂量依赖性。结论rMpf能进入细胞质和细胞核,并且抑制胞内ROS水平、破坏溶酶体最终导致巨噬细胞功能受损。本研究为进一步了解创伤弧菌溶细胞素及类似膜成孔毒素的细胞毒性结构域功能和分子致病机制提供了实验依据。Objective To investigate the cytotoxicity of a recombinant Vibrio vulnificus cytolysin membrane pore-forming peptide(rMpf)to J774A.1 cells as well as its influences on reactive oxygen species(ROS)and lysosomes,and to analyze the cellular localization of the rMpf.Methods The rMpf was expressed using an Escherichia coli expression system and then used to treat murine J774A.1 cells in vitro.The viability of rMpf-and PBS-treated J774A.1 cells and the levels of ROS and lysosomes in these cells were analyzed by flow cytometry.Confocal microscopy was used to observe the cellular localization of rMpf in the J774A.1 cells.Results No significant differences in cell viability,ROS production or lysosome formation in J774A.1 cells were found between low-dose(4μg/ml)rMpf-treated and untreated groups.However,the cell viability,ROS production and lysosome formation were significantly decreased after treating J774A.1 cells with higher doses of rMpf(20 and 60μg/ml).Moreover,the rMpf was found to localize in both the cytoplasm and nuclei of J774A.1 cells in a dose-dependent manner.Conclusions The rMpf could localize in both the cytoplasm and nuclei of J774A.1 cells.It would inhibit the cellular ROS production and lysosome formation to damage macrophage function.This study provided a novel sight for understanding the cytotoxic domain and pathogenesis of Vibrio vulnificus cytolysin and other membrane pore-forming cytolysins.

关 键 词：创伤弧菌 溶细胞素膜成孔肽 活性氧簇 溶酶体 细胞活性 

分 类 号：R378.3[医药卫生—病原生物学]