L929细胞上清诱导小鼠骨髓巨噬细胞脓毒症耐受模型的建立与鉴定  

Establishment and characterization of a sepsis tolerance model in murine bone marrow macrophages induced by L929 cell culture supernatant

作　　者：刘思佳 张亮 刘鑫 蒋青松[1,2] 杨婷 时新惠 钟佩伶 袁烈 黄施群 李小丽 LIU Sijia;ZHANG Liang;LIU Xin;JIANG Qingsong;YANG Ting;SHI Xinhui;ZHONG Peiling;YUAN Lie;HUANG Shiqun;LI Xiaoli(Department of Pharmacology,College of Pharmacy,Chongqing Medical University,Chongqing 400016,China;Chongqing Key Laboratory of Drug Metabolism,Chongqing 400016,China;Medical Research Center,Southwest Hospital,Army Medical University,Chongqing 400038,PR China)

机构地区：[1]重庆医科大学药学院药理系,400016 [2]重庆医科大学药学院药物代谢研究重庆重点实验室,400016 [3]陆军军医大学西南医院临床研究中心,重庆400038

出　　处：《免疫学杂志》2023年第8期654-661,共8页Immunological Journal

基　　金：国家自然科学基金(81873955,82172133);重庆市自然科学基金(csct2020jcyj-msxmX0223 and csct2022nscqmsx0214)。

摘　　要：目的采用L929细胞培养上清分化小鼠骨髓巨噬细胞(BMDMs)建立脓毒症脂多糖(LPS)耐受模型。方法收集L929细胞培养上清,诱导BMDMs分化成熟后,使用显微镜观察BMDMs形态,流式细胞仪鉴定BMDMs纯度及成熟度。采用5 ng/ml LPS预处理分化成熟的BMDMs 4 h后,使用100 ng/ml LPS再处理6 h以建立脓毒症LPS耐受模型。采用ELISA法检测BMDMs耐受模型细胞上清中IL-6及TNF-α释放水平;利用q RT-PCR和Western blot方法检测BMDMs耐受模型细胞中IL-6、TNF-α、TLR4、MyD88和NF-κB p65 m RNA和蛋白表达。结果研究结果表明L929上清中M-CSF因子在第6天释放水平最高;L929上清诱导BMDMs 6 d后细胞分化成熟呈梭形,加入M-CSF阻断剂后细胞未分化;使用F4/80标记分化成熟的BMDMs,阳性率为96.9%;LPS能够剂量及时间依赖性增加分化成熟的BMDMs中IL-6及TNF-α释放水平;在BMDMs、小鼠腹腔巨噬细胞(PMs)、RAW264.7细胞LPS耐受模型中,其耐受组的IL-6及TNF-α释放水平均有不同程度的降低;qRT-PCR及Western blot实验结果均表明在耐受组中IL-6、TNF-α、TLR4、MyD88、NF-κB表达水平显著下调。结论本研究利用L929上清成功诱导BMDMs分化,并建立LPS耐受模型,且LPS诱导BMDMs建立的耐受模型是长期实验的最佳模型,此研究为脓毒症免疫抑制作用及机制提供了理想的细胞模型。This study was designed to establish a sepsis lipopolysaccharide(LPS)tolerance model by using L929 cells.Culture supernatant of L929 cells was collected and induced for preparing bone marrow-derived macrophages(BMDMs),which were then identified by microscopy and flow cytometry in terms of morphology,purity and maturity.After pretreatment of 5 ng/ml LPS for 4 hours,mature BMDMs were treated with 100 ng/ml LPS for 6 hours to establish sepsis LPS tolerance model.IL-6 and TNF-αrelease levels were measured in the supernatant of BMDMs tolerant model cells by ELISA;the mRNA and protein expression of IL-6,TNF-α,TLR4,MyD88 and NF-κB p65 were detected by qRT-PCR and Western blotting.Data showed that macrophage colony stimulating factor(M-CSF)in the L929 supernatant was reached the highest level on the sixth day;mature BMDMs with spindle shape were differentiated in L929 supernatant after 6 days induction,and M-CSF blocking agent could block this differentiation;flow cytometry indicated that the purity of mature BMDMs was 96.9%.LPS could upregulate the release levels of IL-6 and TNF-αfrom mature BMDMs in a dose-and time-dependent manner;In all three different macrophage LPS tolerance models,the IL-6 and TNF-αrelease levels in the tolerance group were reduced,the expression levels of IL-6,TNF-α,TLR4,MyD88,and NF-κB were significantly downregulated.Taken together,L929 supernatant has been used to successfully induce BMDMs differentiation and establish a LPS tolerance model,and the tolerance model established by LPS induction of BMDMs is the best model for long-term experiments,which provides an ideal cell model for the research of the immunosuppressive effect and mechanism of sepsis.

关 键 词：脓毒症 免疫抑制 内毒素耐受 巨噬细胞 骨髓巨噬细胞 

分 类 号：R392-3[医药卫生—免疫学]