诱导C57BL/6小鼠骨髓单核细胞向破骨细胞分化的条件

The conditions of inducing C57BL/6 mouse bone marrow-derived macrophages differentiation into osteoclasts

目的研究诱导C57BL/6小鼠骨髓单核细胞(bone marrow-derived macrophages,BMMs)向破骨细胞分化的条件,提高诱导破骨细胞的效率。方法采用流式细胞仪鉴定BMMs表面标记物,巨噬细胞集落刺激因子(macrophage colonystimulating factor,M-CSF)与核因子κB受体活化因子配体(receptor activator of nuclear factorκB ligand,RANKL)共同诱导BMMs,4天后形成破骨细胞,通过抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)与鬼笔环肽染色鉴定诱导出的破骨细胞,通过骨板吸收实验鉴定破骨细胞的骨吸收活性。结果流式细胞仪检测BMMs的CD11b阳性率为98.4%。经过4天诱导,BMMs激活、融合产生了大量的破骨细胞,呈TRAP阳性,鬼笔环肽染色可见纤维肌动蛋白环形成,骨板上形成大量不规则的骨吸收瘢痕。结论 RANKL诱导BMMs 4天可产生骨吸收活性较高的破骨细胞。

Objective To investigate the conditions inducing C57BL/6 mouse bone marrow-derived macrophages(BMMs)differentiation into osteoclasts,meanwhile increasing the efficiency of osteoclast induced from BMMs.Method Flow cytometer was used to detect the CD11b located in BMMs.The osteoclasts were induced form BMMs with macrophage colony-stimulating factor(M-CSF)and receptor activator of nuclear factorκB ligand(RANKL)for 4 days.And the osteoclasts were confirmed by tartrateresistant acid phosphatase(TRAP)staining and phalloidin staining.The bone absorbent capacity of osteoclasts was measured by bone plate absorption test.Result The CD11b positive incidence detected by flow cytometer of BMMs was 98.4%.After 4 days inducing,BMMs turn into many osteoclasts by activating and fusing,which the TRAP staining was positive and phalloidin staining detect the fibrous actin ring.By means of microscope,a lot of anomalous bone resorption pits were found.Conclusion The osteoclasts that have powerful bone absorbent capacity are differentiated from BMMs with 4 days induction by RANKL.