体外诱导小鼠单核细胞向Kupffer细胞分化的方法建立及特征鉴定  

Establishment and characterization of a method for inducing differentiation of mouse monocyte into Kupffer cells in vitro

作　　者：李伟阳[1] 李丽英[1] 杨琳[1] Li Weiyang;Li Liying;Yang Lin(Department of Cell Biology,Laboratory for Clinical Medicine,School of Basic Medical Sciences,Capital Medical University,Beijing 100069,China)

机构地区：[1]首都医科大学基础医学院细胞生物学系/首都医科大学肝脏免疫与器官保护联合实验室,北京100069

出　　处：《首都医科大学学报》2025年第2期289-295,共7页Journal of Capital Medical University

基　　金：国家自然科学基金项目(82170622);国家资助博士后研究人员计划项目(GZC20231756)。

摘　　要：目的建立一种体外诱导小鼠骨髓单核细胞向Kupffer细胞分化的方法,以用于Kupffer细胞的体外研究。方法从小鼠骨髓中分离单核细胞,经过巨噬细胞集落刺激因子(macrophage colony-stimulating factor,M-CSF)、转化生长因子-β1(transforming growth factor 1,TGF-β1)和δ样蛋白4(delta-like protein 4,DLL4)联合诱导后,采用实时荧光定量聚合酶链反应(real-time fluorescent quantitative polymerase chain reaction,qPCR)法检测骨髓单核细胞来源Kupffer细胞(induced monocyte-derived Kupffer cells,iMoKCs)中表面标志物C型凝集素结构域家族4F(C-type lectin domain family 4,member f,CLEC4F)、C型凝集素结构域家族1B(C-type lectin domain family 1,member b,CLEC1B)和含免疫球蛋白结构域蛋白4(V-set and immunoglobulin domain containing 4,VSIG4)mRNA水平的表达情况;通过免疫荧光的方法检测iMoKCs中蛋白质CLEC4F的表达;利用流式细胞分析技术评估iMoKCs的吞噬功能,用细胞计数试剂盒8(cell counting kit-8,CCK-8)检测iMoKCs的增殖活性。结果从小鼠骨髓中分离的单核细胞,经过24 h的联合诱导,在mRNA水平即可表达Clec4f、Clec1b和Vsig4,并表达蛋白质CLEC4F,流式细胞术分析显示吞噬生物颗粒的iMoKCs约占总细胞数量的80%,CCK-8检测在第24 h、48 h、72 h,iMoKCs数量分别为初始数量(0 h)的1.4、2.0和2.9倍。结论M-CSF、TGF-β1和DLL4联合诱导可在体外成功将骨髓单核细胞分化成为iMoKCs,并且具有吞噬与增殖的能力,可替代原代Kupffer细胞进行体外研究。Objective To establish a method for inducing mouse bone marrow monocytes differentiating into Kupffer cells in vitro,for the study of Kupffer cells.Methods Monocytes were separated from mouse bone marrow and induced by transforming growth factor 1(TGF-β1),macrophage colony-stimulating factor(M-CSF)and delta-like protein 4(DLL4).Induced monocyte-derived Kupffer cells(iMoKCs)were identified by real-time fluorescent quantitative polymerase chain reaction(qPCR),immunofluorescence staining and flow cytometry.Immunofluorescence staining and flow cytometry were used to evaluate the phagocytic capacity of iMoKCs.Cell counting kit-8(CCK-8)was used to evaluate the proliferation capacity.Results Bone marrow monocytes were isolated,after 24 h combined induction,iMoKCs express C-type lectin domain family 4,member f(CLEC4F),C-type lectin domain family 1,member b(CLEC1B)and V-set and immunoglobulin domain containing 4(VSIG4)at mRNA level,and protein CLEC4F.Phagocytic function of iMoKCs were detected by flow cytometry,and nearly 80%of total iMoKCs show bioparticle acceptance.The number of iMoKCs were detected by CCK-8,the counts were 1.4,2.0 and 2.9 times of the initial number(0 h)at 24,48 and 72 h.Conclusion These cells display the immunophenotype of Kupffer cells and show the ability of phagocytosis and proliferation induction through M-CSF,TGF-β1 and DLL4,which could replace primary Kupffer cells for in vitro research.

关 键 词：小鼠 KUPFFER细胞 吞噬 增殖 体外诱导 单核细胞 

分 类 号：Q2[生物学—细胞生物学]