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

背景：目前骨髓单核细胞的分离提取国内外文献报道，大多是利用RAW264.7细胞诱导分化为破骨细胞。 目的：探讨分离、培养、纯化和鉴定C57BL/6小鼠骨髓单核细胞的方法，观察小鼠骨髓单核细胞体外生长特征，诱导其向破骨细胞分化。 方法：无菌分离C57BL/6小鼠股骨和胫骨，取出骨髓细胞，提取过程中先用红细胞裂解液去除红细胞；骨髓细胞过夜培养后(大于16 h)，第2天分离出悬浮细胞，在含有巨噬细胞集落刺激因子的培养基中继续培养获得贴壁的小鼠骨髓单核细胞。通过换液对小鼠骨髓单核细胞进行纯化和扩增培养。进行形态学观察，测定生长曲线，用流式细胞仪检测原代小鼠骨髓单核细胞细胞表面抗原，加入巨噬细胞集落刺激因子和核因子κB受体活化因子配基诱导分化为破骨细胞。 结果与结论：新分离的小鼠骨髓单核细胞呈小圆形，两端伸出触角，培养5 d后，细胞成椭圆形，两端的触角更明显，增殖依赖巨噬细胞集落刺激因子。流式结果显示分离的小鼠骨髓单核细胞纯度较高，能够诱导分化为破骨细胞。利用间充质干细胞与单核细胞的贴壁能力不同及巨噬细胞集落刺激因子显著促进单核细胞贴壁增殖的特性能有效分离获得稳定生长的小鼠骨髓单核细胞。培养的小鼠骨髓单核细胞性状稳定，表型稳定均一，适于做进一步研究。中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程全文链接：

C57BL/6 mouse bone marrow monocytes: isolation,cultivation, purification and differentiation into osteoclasts

BACKGROUND: Isolation and extraction of bone marrow monocytes is mostly realized via osteoclast differentiation induced by RAW264.7 cells 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 monocytes in vitro. METHODS: The femurs and tibias of C57BL/6 mice were aseptically removed to extract bone marrow cells that were cultured overnight (> 16 hours). During the extraction process, erythrocyte lysate was used to remove red blood cells. At day 2, suspension cells 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. Cell morphology was observed, the growth curve was tested and using flow cytometry, the cell 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 cells into osteoclasts.    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 cells were oval-shaped with more obvious feelers, and cell 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 cells and monocytes as well as macrophage colony-stimulating factor. Cultured mouse bone marrow monocytes are phenotypically stable and can be used for further research.