刚地弓形虫热休克蛋白21体外条件下对小鼠巨噬细胞功能的影响  

Effects of Toxoplasma gondii heat shock protein 21 on the function of mouse macrophage in vitro

作　　者：李纯静 周天缘 于正青 储稳 徐立新[1] 宋小凯[1] 严若峰[1] 李祥瑞[1] LI Chunjing;ZHOU Tianyuan;YU Zhengqing;CHU Wen;XU Lixin;SONG Xiaokai;YAN Ruofeng;LI Xiangrui(College of Veterinary Medicine/MOE Joint International Research Laboratory of Animal Health and Food Safety,Nanjng Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学动物医学院/教育部动物卫生与食品安全国际联合研究实验室,江苏南京210095

出　　处：《南京农业大学学报》2021年第1期136-143,共8页Journal of Nanjing Agricultural University

基　　金：家畜疫病病原生物学国家重点实验室(中国农业科学院兰州兽医研究所)开放课题资助;江苏省高等学校重点学科项目(PAPD)。

摘　　要：[目的]本试验旨在探究刚地弓形虫热休克蛋白21(TgHSP21)在体外对小鼠巨噬细胞系(Ana-1)功能的调节作用。[方法]构建重组表达载体pET-32a/TgHSP 21,获得重组蛋白TgHSP21(rTgHSP21)。用免疫荧光抗体试验(IFA)验证rTgHSP21在体外与Ana-1细胞的结合情况。Ana-1细胞分别用不同质量浓度(5、10、20、40、80μg·mL^-1)的rTgHSP21处理后,采取细胞计数试剂盒(CCK-8)检测细胞增殖情况,应用Transwell细胞小室检测细胞趋化情况,应用流式细胞术检测Ana-1细胞凋亡和吞噬能力。分别用总一氧化氮(NO)试剂盒和细胞因子ELISA试剂盒检测Ana-1细胞的NO和肿瘤坏死因子α(TNF-α)、白介素12(IL-12)、IL-6、IL-1β的分泌量。[结果]成功获得纯化的rTgHSP21,其具有与Ana-1细胞结合的能力。5和10μg·mL^-1的rTgHSP21均显著促进Ana-1细胞的增殖,各个浓度的rTgHSP21均显著促进细胞凋亡和吞噬能力。80μg·mL^-1 rTgHSP21明显促进Ana-1细胞分泌NO。在不同浓度rTgHSP21作用下,细胞因子IL-6和TNF-α的表达量均明显上升,而其质量浓度为10、20、40和80μg·mL^-1时IL-12的分泌被抑制。此外,rTgHSP21显著抑制了Ana-1细胞的迁移能力。[结论]TgHSP21能够与Ana-1细胞结合并在体外条件下调节Ana-1细胞的增殖、吞噬、凋亡、迁移及分泌。[Objectives]The aim of this study was to investigate the effects of Toxoplasma gondii heat shock protein 21(TgHSP21)on the function of mouse macrophage(Ana-1)in vitro.[Methods]The recombinant expression vector pET-32a/TgHSP 21 was constru-cted and the recombinant protein TgHSP21(rTgHSP21)was obtained.The immunofluorescence assay(IFA)was used to verify the ability of rTgHSP21 to bind to Ana-1 cells.Following treatment with different concentrations(5,10,20,40 and 80μg·mL^-1)of rTgHSP21,the proliferation of Ana-1 cells was evaluated by cell counting kit(CCK-8)and the Ana-1 cells migration was measured by Transwell system,and then the effects of apoptosis and phagocytosis of Ana-1 cells were calculated by flow cytometry.The secretion of nitric oxide(NO),Tumor necrosis factorα(TNF-α),interleukin 12(IL-12),interleukin 6(IL-6)and interleukin 1β(IL-1β)were separately analyzed by total nitric oxide assay kit and mouse cytokine ELISA kit.[Results]The purified rTgHSP21 was successfully obtained,and had the ability to bind to Ana-1 cells.The proliferation of Ana-1 cells was significantly promoted by 5 and 10μg·mL^-1 concentration of rTgHSP21.Both of the apoptosis and the phagocytic capacity of Ana-1 cells were significantly enhanced by every concentration.The section of NO in Ana-1 cells was increased at 80μg·mL^-1 rTgHSP21.The expressions of IL-6 and TNF-αwere increased significantly at different concentrations,however,the expression of IL-12 was inhibited at concentrations of 10,20,40 and 80μg·mL^-1 rTgHSP21.In addition,the migration ability of Ana-1 cells was also significantly inhibited.[Conclusions]TgHSP21 could bind to Ana-1 cells and regulate the proliferation,phagocytosis,apoptosis,migration and secretion functions of Ana-1 cells in vitro.

关 键 词：弓形虫 热休克蛋白21 小鼠巨噬细胞系 免疫功能 

分 类 号：S855.9[农业科学—临床兽医学]