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.
作者
刘思佳
张亮
刘鑫
蒋青松
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)
出处
《免疫学杂志》
CAS
CSCD
北大核心
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)。
