LPS与ATP共同诱导小鼠原代腹腔巨噬细胞焦亡模型的建立  

Establishment of pyroptosis model on primary peritoneal macrophages induced by LPS and ATP

作　　者：刘慧玲 吴传新[1,2] 龙贤梨 李丽[1] 李飞[1] 郭晖[1] 孙航(指导)[1] LIU Huiling;WU Chuanxin;LONG Xianli;LI Li;LI Fei;GUO Hui;SUN Hang(Institute for Viral Hepatitis,the Second Affiliated Hospital,Chongqing Medical University,Chongqing 400010,China)

机构地区：[1]重庆医科大学附属第二医院病毒性肝炎研究所,重庆400010 [2]重庆医科大学附属第二医院肝胆外科,重庆400010

出　　处：《中国免疫学杂志》2023年第10期2028-2033,共6页Chinese Journal of Immunology

基　　金：国家自然科学基金面上项目(81871608)。

摘　　要：目的:探索脂多糖(LPS)和三磷酸腺苷(ATP)共同诱导小鼠原代腹腔巨噬细胞焦亡模型的最佳条件。方法:采用流式细胞仪F4/80和CD-11b染色检测巨噬细胞纯度,Annexin V-PE/7-AAD双染色法筛选出LPS和ATP共同诱导细胞焦亡的最适浓度及时间。巨噬细胞随机分为control组、LPS组、ATP组和LPS+ATP组;Western blot检测GSDMD、caspase-1、caspase-11、NLRP3、ASC、pro-IL-1β、pro-IL-18和HMGB1蛋白表达水平;ELISA检测培养上清中IL-1β和TNF-α表达水平;透射电镜(TEM)和扫描电镜(SEM)观察巨噬细胞焦亡形态。结果:巨噬细胞的纯度达到90%;500 ng/ml LPS 24 h+5 mmol/L ATP 4 h为诱导巨噬细胞焦亡的最佳组合方式;LPS+ATP组的GSDMD、caspase-1、caspase-11、NLRP3、ASC、pro-IL-1β、pro-IL-18和HMGB1的蛋白表达量明显高于对照组(P<0.05);培养上清中IL-1β和TNF-α表达量显著高于对照组(P<0.05);电镜下可观察到明显的焦亡特征。结论:成功建立了LPS和ATP共同诱导小鼠原代腹腔巨噬细胞的焦亡模型,为深入探讨免疫细胞焦亡的分子机制提供了稳定的细胞模型。Objective:To explore optimal condition of a model of pyroptosis on primary peritoneal macrophages induced by the lipopolysaccharide(LPS)and adenosine triphosphate(ATP).Methods:Purity of macrophages was detected by flow cytometric with F4/80 and CD11-b,and Annexin V-PE/7-AAD double staining was used to detect pyroptosis cell for screening the optimum concentration and time of pyroptotic cells induced by LPS and ATP.Macrophages were randomly divided into control group,LPS group,ATP group and LPS+ATP group.Expressions of GSDMD,caspase-1,caspase-11,NLRP3,ASC,pro-IL-1β,pro-IL-18 and HMGB1 proteins were detected by Western blot.Levels of IL-1βand TNF-αin culture supernatant were measured by ELISA.Structure of pyroptosis macrophages was observed by transmission electron microscope(TEM)and scan electron microscope(SEM).Results:Purity of primary peritoneal macrophages could be 90%;500 ng/ml LPS 24 h and 5 mmol/L ATP 4 h was the optimal combination of inducing macrophages pyroptosis.Compared with control group,LPS and ATP group had significantly increased protein expressions of GSDMD,caspase-1,caspase-11,NLRP3,ASC,pro-IL-1β,pro-IL-18 and HMGB1(P<0.05),and levels of IL-1βand TNF-αin culture supernatant were significantly higher than that in control group(P<0.05);structure of pyroptosis macrophages could be obviously observed by TEM and SEM.Conclusion:Pyroptosis model of primary peritoneal macrophages induced by LPS and ATP is successfully established,which provides a cell model for exploring the molecular mechanism of pyroptosis on immune cells in the future.

关 键 词：LPS ATP 细胞焦亡 原代腹腔巨噬细胞 脓毒症 

分 类 号：R392.1[医药卫生—免疫学]