小鼠骨髓巨噬细胞体外培养及增殖情况的探索  

In-vitro culture method and proliferation of mouse bone marrow-derived macrophage

作　　者：祝文 崔凤 程志坚[2] 贺西京[2] ZHU Wen;CUI Feng;CHENG Zhijian;HE Xijing(Deparment of Critical Care Medicine,the First People′s Hospital of Xianyang,Xianyang,Shaanxi 710021,China;Department of Orthopaedics,the Second Affiliated Hospital of Medical College of Xi′an Jiaotong University,Xi′an,Shaanxi 710004,China)

机构地区：[1]陕西省咸阳市第一人民医院重症医学科,710021 [2]西安交通大学医学院第二附属医院骨科,710004

出　　处：《重庆医学》2019年第17期2909-2912,共4页Chongqing medicine

基　　金：国家自然科学基金项目(81801237,81571209);陕西省自然科学基础研究计划项目(2018JQ8039)

摘　　要：目的建立小鼠骨髓来源巨噬细胞的体外培养方法及其标志物表达、增殖情况,为相关研究提供理论基础。方法从6~8周龄昆明小鼠股骨分离得到骨髓单细胞悬液,在含15%L929上清液的培养液中培养。在1、3、5、7 d不同时间点,利用免疫荧光染色检测其巨噬细胞标志物F4/80表达情况,同时利用Edu标记法检测细胞增殖情况。结果用含L929上清液的培养液诱导培养骨髓细胞1、3、5、7 d后细胞F4/80阳性细胞率分别为(1.52±0.39)%、(17.95±2.36)%、(75.57±6.17)%、(95.49±9.83)%,随着培养时间延长,F4/80阳性细胞率逐渐升高,培养骨髓细胞7 d后吞噬能力明显,不同培养时间点F4/80阳性细胞率比较差异均有统计学意义(1 d vs.3 d、3 d vs.5 d、5 d vs.7 d,P<0.05)。随着培养时间延长Edu阳性细胞逐渐增高,培养1、3、5、7 d后细胞EdU阳性细胞率分别为(2.13±0.33)%、(4.61±0.54)%、(18.02±2.19)%、(28.84±3.27)%,不同培养时间点EdU阳性细胞率比较,差异均有统计学意义(1 d vs.3 d、3 d vs.5 d、5 d vs.7 d,P<0.05)。结论体外培养可成功获得小鼠骨髓来源巨噬细胞,培养7 d是获得高纯度巨噬细胞的重要条件。Objective To establish a culture method for mouse bone marrow-derived macrophages and to observe the expression of proliferation markers in ovder to provide theoretical basis for relevant research.Methods Bone marrow mono cells were isolated from femur of 6-8 weeks Kunming mice and cultured in the medium containing 15%L929 supernatant.The expression of macrophage marker F4/80 was detected by immunofluorescence staining and the proliferation was tested by EdU labeling at different time points(1,3,5 and 7 d).Results After 1,3,5 and 7 days of in-vitro culture in medium with L929 supernatant,the F4/80 positive rate in bone marrow cells was(1.52±0.39)%,(17.95±2.36)%,(75.57±6.17)%,and(95.49±9.83)%,respectively.With the extension of culture time,the positive rate of F4/80 gradually increased,and the phagocytic capacity of bone marrow cells cultured for 7 d was obvious.The differences of F4/80 positive rate among different time points were statistically significant(1 d vs.3 d,3 d vs.5 d,and 5 d vs.7 d,P<0.05).The rates of EdU positive cells in bone marrow cells were(2.13±0.33)%,(4.61±0.54)%,(18.02±2.19)%,and(28.84±3.27)%after 1,3,5,and 7 days of culture,respectively.The difference of EdU positive cell rate among groups were statistically significant(1 d vs.3 d,3 d vs.5 d,and 5 d vs.7 d,P<0.05).Conclusion Bone marrow derived macrophages can be successfully cultured in vitro,culture for 7days is an important condition for obtaining high purity macrophages.

关 键 词：巨噬细胞 细胞培养技术 细胞增殖 

分 类 号：R392[医药卫生—免疫学]