铁调素对小鼠单核细胞RAW264.7向破骨细胞分化的影响

Effects of hepcidin treatment on differentiation of mouse RAW264.7 monocytes into osteoclasts

目的探讨铁调素对小鼠单核细胞株RAW264．7向成熟破骨细胞分化的影响。方法将不同浓度铁调素加入含有核因子KB受体活化因子配体（RANKL）的RAW264．7细胞培养基，24h后用CCK-8法检测细胞的增生活性；4d后对细胞进行抗酒石酸酸性磷酸酶（TRAP）染色，光镜下观察；用RT—PCR法检测TRAP、组织蛋白酶K（CTK）、金属蛋白酶9（MMM-9）基因表达；用酶联免疫吸附法（ELISA）检测上清液中TRAP-5b含量；24h后用共聚焦显微镜（CLSM）测定细胞内铁离子浓度。结果铁调素在0～800nmol／L浓度围内可增加RAW264.7细胞TRAP染色阳性数目，上调TRAP、CTK和MMP-9基因表达，增加上清液TRAP-Sb含量（P〈0．05），同时增加RAW264．7细胞内铁离子浓度（P〈0．05）。结论铁调素可以促进RAW264．7细胞向破骨细胞分化，其作用机制可能与铁调素增加细胞内铁离子有关。

Objective To investigate the effects of hepcidin on differentiation of mouse RAW264.7 monocytes into osteoclasts. Methods RAW264. 7 cells were treated with different concentrations of bepcidin in the presence of receptor activator of NF-Kb ligand （RANKL）. Cell viability, the number of tartrate-resistant acid phosphatase （TRAP） - positive cells, levels of TRAP, cathepsin K （CTK） , and matrix metalloproteinase 9 （MMP-9） mRNA, and levels of TRAP-Sb protein in the supernatant were examined. The intracellular iron ion was measured by a confocal laser scanning microscope. Results Hepcidin at 0-800 nmol/1 could significantly increase the number of TRAP-positive MNCs, and up- regulate gene expression of TRAP, CTK, and MMP-9, and increase the concentration of TRAP-5b in the supernatant, and increase concentrations of intracellular iron of RANKL-induced RAW264. 7 cells （P 〈 0. 05 ）. Conclusion Hepcidin can significantly facilitate RANKL-induced differentiation of RAW264.7 into osteoclasts in vitro. The mechanism behind accelerated differentiation involves increased levels of intracellular iron.