破骨细胞分化因子诱导小鼠骨髓细胞形成破骨细胞的实验研究
Osteoclast Formation Induced by Osteoclast Differentiation Factor in Primary Murine Marrow Cell Culture
作者：郭子杰,栾文民,于世凤,张铁梅
Author：Guo Zijie, Iuan Wenmin, Yu Shifeng, Zhang Tiemei
收稿日期：2002-12-25 年卷（期）页码：2002,20(06):445-
期刊名称：华西口腔医学杂志
Journal Name：West China Journal of Stomatology
关键字：破骨细胞分化因子,巨噬细胞集落刺激因子,破骨细胞,小鼠,骨髓细胞,
Key words：Osteoelast differentiation factor,macrophage colony-stimulating factor osteoclast,mice,bone marrow cell,
基金项目：国家自然科学基金资助项目(编号 39830430)
中文摘要
目的:探讨破骨细胞分化因子(ODF)和巨噬细胞集落刺激因子(M-CSF)联合应用进行体外诱导小鼠骨髓细胞形成破骨细胞(OC)的能力。方法:收集5～6周小鼠骨髓细胞,在含M-CSF(10 ng/ml)的α-MEM全培养液中培养24h,然后将悬浮生长的细胞接种到24孔培养板,并加入不同浓度的ODF和M-CSF。,通过观察抗酒石酸盐酸性磷酸酶(TRAP)染色的阳性细胞和能否在骨磨片上形成吸收陷窝来鉴定OC形成情况。结果:在只加入ODF或M-CSF一种细胞因子时,未见有TRAP阳性或CTR阳性细胞形成,同时加入ODF和M-CSF,可形成典型的OC。TRAP阳性多核细胞的数目和培养液中钙离子浓度的增加与ODF的浓度呈正相关。结论:小鼠骨髓来源的单核细胞在ODF和M-CSF共同作用下可形成具有骨吸收功能的OC,为体外研究OC的分化发育和功能调节提供了一种新的方法。
英文摘要
Objective: Both ODF (osteoclast differentiation factor, a newly identified member of the tumor necrosis factor super-family) and M-CSF (macrophage colony-stimulating factor) are indispensable for OC (osteoclast) formation. The purpose of this study was to test the possibility that the combination of ODF and M-CSF was sufficient for OC development in primary murine marrow cell culture. Methods: Bone marrow cells were isolated from 5- to 6- week-old mice and incubated in M-CSF (10 ng/ml). After 24 hours, non-adherent cells were harvested and resuspended in a-MEM /BCS. The suspension was added to the wells of 24-well plates with different concentration of ODF and/or M-CSF. TRAP (tartrate-resistant acid phosphatase) staining was used to identify OC. The bone resorption pits on slices of bovine cortical bone were examined with inverted phase contrast microscope, and the changes of Ca2+ concentration in the medium during whole culture period were measured by atomic absorption spectrophotometer. Results: When bone marrow cells were cultured for up to 11 days either in the presence of M-CSF (10 ng/ml) or ODF (50 ng/ml) , no cells expressing TRAP and bone obvious resorption was found. But in the presence of ODF and M-CSF, many TRAP-positive mono- and multi-nucleated cells were formed after 7 days. The number of TRAP-positive miJti-nucleated cells and the change of Ca2+ in the culture medium increased dose-dependently with ODF concentration. Conclusion: The combination of ODF and M-CSF can induce OC formation and bone resorption in murine marrow cell culture, and it can be employed to investigate the direct effects of factors on OC differentiation and activation.
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