中国组织工程研究Issue(6)：940-944,5.DOI:10.3969/j.issn.2095-4344
C57BL/6小鼠骨髓单核细胞分离、培养、纯化及向破骨细胞的分化
C57BL/6 mouse bone marrow monocytes：isolation, cultivation, purification and differentiation into osteoclasts
周龙 1陈曦 2罗宗平 3杨惠林 1何帆2
作者信息
- 1. 苏州大学附属第一人民医院骨科，江苏省苏州市 215006
- 2. 苏州大学骨科研究所，江苏省苏州市 215007
- 3. 苏州大学骨科研究所，江苏省苏州市 215007
- 折叠
摘要
Abstract
BACKGROUND:Isolation and extraction of bone marrow monocytes is mostly realizedvia osteoclast differentiation induced by RAW264.7 cels as reported at home and abroad. OBJECTIVE:To explore a new method for the isolation, culture, purification and identification of C57BL/6 mouse bone marrow monocytes and to observe the growth features and osteoclast differentiation of mouse bone marrow monocytesin vitro. METHODS: The femurs and tibias of C57BL/6 mice were asepticaly removed to extract bone marrow cels that were cultured overnight (> 16 hours). During the extraction process, erythrocyte lysate was used to remove red blood cels. At day 2, suspension cels were isolated and cultured with the medium containing macrophage colony-stimulating factor to harvest adhered mouse bone marrow monocytes. The medium was changed to purify and amplify the mouse bone marrow monocytes. Cel morphology was observed, the growth curve was tested and using flow cytometry, the cel surface antigen of primary mouse bone marrow monocytes was detected. Macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand were added to differentiate the cels into osteoclasts.
<br> RESULTS AND CONCLUSION: The newly separated mouse bone marrow monocytes were circular and feelers were extended from both ends. After 5 days of culture, the cels were oval-shaped with more obvious feelers, and cel proliferation relied on macrophage colony-stimulating factor. Under the flow cytometry, isolated mouse bone marrow monocytes had high purity and could be induced to differentiate into osteoclasts. It is effective to isolate mouse bone marrow monocytes that grow stably by vigorously promoting the proliferation of monocytes by using different adherent abilities of mesenchymal stem cels and monocytes as wel as macrophage colony-stimulating factor. Cultured mouse bone marrow monocytes are phenotypicaly stable and can be used for further research.关键词
干细胞/培养/骨髓单核细胞/破骨细胞/巨噬细胞集落刺激因子/核因子κB受体活化因子配基/鉴定/分化/药物贴壁筛选法Key words
Subject headings:Monocytes/Osteoclasts/Macrophage Colony-Stimulating Factor/RANK Ligand分类
医药卫生引用本文复制引用
周龙,陈曦,罗宗平,杨惠林,何帆..C57BL/6小鼠骨髓单核细胞分离、培养、纯化及向破骨细胞的分化[J].中国组织工程研究,2015,(6):940-944,5.
