小鼠骨髓来源巨噬细胞培养模型的建立及鉴定  

Establishment and identification of bone marrow-derived macrophage culture model

作　　者：马玲玲 马红梅 朱源鹤 宋孚洋 史客松[1,2] 马臣杰 曾瑾 MA Ling-ling;MA Hong-mei;ZHU Yuan-he;SONG Fu-yang;SHI Ke-song;MA Chen-jie;ZENG Jin(Key Laboratory of Ministry of Education for Conservation and Utilization of Special Biological Resources in the Western,Ningxia University,Yinchuan 750021,Ningxia Hui Autonomous Region,China;不详)

机构地区：[1]宁夏大学西部特色生物资源保护与利用教育部重点实验室,宁夏银川750021 [2]宁夏大学生命科学学院,宁夏银川750021

出　　处：《中国生物制品学杂志》2022年第6期706-710,717,共6页Chinese Journal of Biologicals

基　　金：国家自然科学基金(31660719);宁夏自然科学基金(2020AAC03073);宁夏回族自治区重点研发计划项目(东西部合作)(2017BN04)。

摘　　要：目的建立小鼠骨髓来源巨噬细胞(bone marrow-derived macrophages,BMDM)培养模型,并对BMDM进行细胞表面标志物及吞噬能力鉴定。方法采用32℃培养10 d和37℃培养3 d的方式培养L929细胞,检测细胞上清中巨噬细胞集落刺激因子(macrophage colony stimulating factor,M-CSF)的浓度。获取小鼠骨髓细胞后,分别以M-CSF和两种条件培养的L929细胞上清进行诱导分化,通过光学显微镜对诱导分化后的BMDM进行形态学观察;流式细胞术检测CD11b阳性细胞株所占百分比,免疫荧光法观察诱导后巨噬细胞表面标志抗原CD11b和CD16的表达;对表达绿色荧光蛋白的大肠埃希菌进行吞噬试验鉴定BMDM的吞噬功能。结果32℃培养10 d的L929细胞上清中M-CSF的浓度显著高于37℃培养3 d(P<0.05)。3种诱导方式均可使小鼠骨髓源细胞分化成熟,经32℃培养10 d的L929细胞上清诱导分化后的BMDM生长状态最佳。流式细胞术检测显示巨噬细胞表面标志抗原CD11b阳性率为80.62%,免疫荧光检测显示诱导分化后的BMDM细胞表面有巨噬细胞标志性抗原CD11b和CD16的表达,且具有对表达绿色荧光蛋白的大肠埃希菌良好的吞噬能力。结论成功制备了小鼠BMDM,并优化了制备和鉴定的条件,为研究免疫细胞功能、病原微生物与巨噬细胞相互作用提供了理想的细胞模型制备方法。Objective To establish mouse bone marrow-derived macrophage(BMDM)culture model and identify the cell surface markers and phagocytic ability.Methods L929 cells were cultured at 32℃for 10 d or at 37℃for 3 d,and the concentration of macrophage colony stimulating factor(M-CSF)in culture supernatant was determined.Mouse bone marrow cells were isolated and induced with M-CSF and L929 cell supernatants at two culture conditions respectively.The morphology of differentiated BMDMs was observed by optical microscopy,while the percentage of CD11b positive cell strains by flow cytometry,and the expressions of surface marker antigens CD11b and CD16 by immunofluorescence assay(IFA).The phagocytic function of macrophages was identified by phagocytosis test on E.coli expressing green fluorescence protein(GFP).Results The M-CSF concentration in supernatant of L929 cells cultured at 32℃for 10 d was significantly higher than that at 37℃for 3 d(P<0.05).All the mouse bone marrow cells were differentiated and matured after induction by three methods.However,the growth state of BMDMs induced with L929 cell supernatant cultured at 32℃for 10 d was satisfactory.Flow cytometry showed that the positive rate of the macrophage surface marker antigen CD11b was 80.62%.IFA showed the expressions of CD11b and CD16 on surface of BMDM after induction and good phagocytosis ability to E.coli expressing GFP.Conclusion Mouse BMDMs were successfully prepared,of which the condition for preparation and identification were optimized,which provided an ideal method for preparation of cell model for study on immune cell function and the interaction between pathogenic microorganisms and macrophages.

关 键 词：小鼠骨髓来源巨噬细胞 表面标志物 吞噬能力 

分 类 号：R392[医药卫生—免疫学]