阴道毛滴虫巨噬细胞迁移抑制因子对THP-1巨噬细胞的作用  

Effect of Trichomonas vaginalis macrophage migration inhibitory factor on THP-1 macrophages

作　　者：林琳 黄霜 张峰源 李家政 姜旭淦 陈盛霞 LIN Lin;HUANG Shuang;ZHANG Fengyuan;LI Jiazheng;JIANG Xugan;CHEN Shengxia(Department of Laboratory Medicine,School of Medicine,Jiangsu University,Zhenjiang,Jiangsu 212013,China;National Insti-tute of Parasitic Diseases,Chinese Center for Disease Control and Prevention(Chinese Center for Tropical Diseases Research),Na-tional Health Commission Key Laboratory of Parasite and Vector Biology,WHO Collaborating Centre for Tropical Diseases,Nation-al Center for International Research on Tropical Diseases,Shanghai 200025,China)

机构地区：[1]江苏大学医学院检验系,江苏镇江212013 [2]中国疾病预防控制中心寄生虫病预防控制所(国家热带病研究中心)、国家卫生健康委员会寄生虫病原与媒介生物学重点实验室、WHO热带病合作中心、国家级热带病国际联合研究中心,上海200025

出　　处：《中国血吸虫病防治杂志》2023年第1期29-37,共9页Chinese Journal of Schistosomiasis Control

基　　金：国家寄生虫资源库建设项目(2019-194-30)。

摘　　要：目的探讨阴道毛滴虫分泌的巨噬细胞迁移抑制因子(TvMIF)对THP-1巨噬细胞的作用。方法原核表达重组TvMIF蛋白并纯化,鉴定后去内毒素。采用不同浓度(0、1、5、10、50 ng/mL和100 ng/mL)重组TvMIF蛋白作用于THP-1巨噬细胞,用细胞计数试剂盒(cell counting kit,CCK)-8检测重组TvMIF蛋白对THP-1巨噬细胞的毒性,采用流式细胞术检测细胞凋亡和活性氧(reactive oxygen species,ROS)水平,采用实时荧光定量PCR技术(real-time fluorescence quantitative PCR,qPCR)检测核苷酸结合寡聚化结构域样受体蛋白(nucleotide-binding oligomerization domain-like receptorprotein 3,NLRP3)、caspase-1、白细胞介素1β(interleukin-1β,IL-1β)、IL-18mRNA表达水平变化,采用Western blotting技术检测THP-1巨噬细胞中caspase1、NLRP3、消化道皮肤素D(gasdermin D,GSDMD)及GSDMD蛋白氨基端裂解片段(gasdermin D N-terminal,GSDMD-NT)、pro-IL-1β蛋白表达水平变化。结果成功表达并纯化获得分子量为15.5 kDa的重组TvMIF蛋白。内毒素检测结果显示,已去除重组TvMIF蛋白的内毒素(浓度<0.1 EU/mL),可用于蛋白功能研究。流式细胞术检测结果显示,当重组TvMIF蛋白浓度≤10 ng/mL时可促进THP-1巨噬细胞凋亡,当TvMIF浓度为5 ng/mL时凋亡现象最为明显,当重组TvMIF蛋白浓度为50、100 ng/mL时可抑制THP-1巨噬细胞凋亡。当TvMIF蛋白浓度为1 ng/mL时,ROS含量明显升高。qPCR检测结果显示,1 ng/mL重组TvMIF蛋白作用THP-1巨噬细胞8 h后,caspase-1、NLRP3、IL-18和IL-1βmRNA水平均显著上升。Western blotting检测结果显示,1 ng/mL重组TvMIF蛋白刺激THP-1巨噬细胞后,caspase-1和NLRP3蛋白表达量显著上升,pro-IL-1β、GSDMD和GSDMD-NT蛋白表达量显著增加;用NLRP3炎症小体特异性抑制剂MCC950预处理后,GSDMD和GSDMD-NT蛋白表达水平显著下降。结论高浓度重组TvMIF蛋白抑制THP-1巨噬细胞凋亡;低浓度重组TvMIF蛋白激活NLRP3炎症小体,促进THP-1巨噬细胞焦亡。Objective To investigate the effect of Trichomonas vaginalis macrophage migration inhibitory factor(TvMIF)on THP-1 macrophages.Methods Recombinant TvMIF protein was prokaryotic expressed and purified,and endotoxin was removed after identification.Following exposure to TvMIF at concentrations of 0,1,5,10,50 and 100 ng/mL,the cytotoxicity of the recombinant TvMIF protein to THP-1 macrophages was tested using cell counting kit(CCK)-8 assay,and the apoptosis of THP-1macrophages and reactive oxygen species(ROS)were detected using flow cytometry.The relative expression of nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3),caspase-1,interleu kin-1β(IL-1β)and IL-18 genes was quantified using real-time fluorescent quantitative PCR(qPCR)assay,and the expression of caspase-1,NLRP3,gasdermin D(GSDMD),gasdermin D N-terminal(GSDMD-NT)and pro-IL-1βproteins were determined using Western blotting assay.Results Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)displayed successful expression and purification of the recombinant TvMIF protein with a molecular weight of 15.5 kDa,and the endotoxin activity assay showed the successful removal of endotoxin in the recombinant TvMIF protein(endotoxin concentration<0.1 EU/mL),which was feasible for the subsequent studies on protein functions.Flow cytometry revealed that the recombinant TvMIF protein at a concentration of 10 ng/mL and less promoted the apoptosis of THP-1 macrophages,and the highest apoptotic rate of THP-1 macrophages was seen following exposure to the recombinant TvMIF protein at a concentration of 5 ng/mL,while the recombinant TvMIF protein at concentrations of 50 and 100 ng/mL inhibited the apoptosis of THP-1 macrophages-Exposure to the recombinant TvMIF protein at a concentration 1 ng/mL resulted in increased ROS levels in THP-1 macrophages.qPCR assay quantified significantly elevated caspase-1,NLRP3,IL-18 and IL-1βexpression in THP-1 macrophages 8 hours post-treatment with the recombinant TvMIF protein at a concentration 1 ng/m

关 键 词：阴道毛滴虫 巨噬细胞迁移抑制因子 THP-1巨噬细胞 NLRP3炎症小体 细胞凋亡 细胞焦亡 

分 类 号：R382.21[医药卫生—医学寄生虫学]