白三烯B4对人破骨细胞直接分化和激活功能的实验研究

The study of direct differentiation and activation effect of leukotriene B4 on human osteoclast

目的探讨白三烯(LT)B4能否不依赖细胞核因子资B受体激活剂配体(RANKL)直接促进人破骨细胞的分化和激活。方法阳性对照组用25ng/ml巨噬细胞集落刺激因子(M-CSF)和30ng/mlsRANKL来诱导人外周血单核细胞的培养,实验组用25ng/mlM-CSF和10-9、10-8、10-7mol/LLTB4来诱导。通过抗酒石酸磷酸酶(TRAP)染色及10mm×10mm玻片上多核性TRAP染色(+)的破骨细胞样细胞计数,来确定LTB4的直接分化作用,并与RANKL的作用比较。通过甲苯胺蓝染色10mm×10mm牛皮质骨片上的骨质吸收陷窝并计数,来确定LTB4直接功能激活作用,并与RANKL的作用比较。结果当M-CSF存在时,LTB4能够直接分化人外周血单核细胞为破骨细胞样细胞,并能激活其骨质吸收功能。且随LTB4浓度的增加而增强,但要弱于RANKL。结论LTB4对人破骨细胞有不依赖RANKL的直接分化和激活作用。

Objective To determine whether leukotriene B4 (LTB4) could directly stimulate human osteoclast differentiation and activation independent of RANKL (ODF). Methods In positive control group, the peripheral blood monocytes were cultured in the presence of 25ng/ml macrophage colony-stimulating factor (M-CSF) and 30 ng/ml sRANKL. In experimental groups, three different concentrations of LTB4 (10-9, 10-8 and 10-7 mol/L) were added to the culture of CD14+ monocyte fraction of peripheral blood mononuclear cell (PBMC) in the presence of M-CSF. Through TRAP staining and counting the number of osteoclast-like cells on 10 mm×10 mm glass coverslips, the direct differentiation effect of LTB4 was confirmed and compared with that of RANKL. Through toluidine blue staining and couning the number of bone resorption lacunae on 10 mm×10 mm bovine cortical slices, the direct activation effect of LTB4 was confirmed and compared with that of RANKL. Results LTB4 could induce multinucleated osteoclast-like cells which were positive for Tartrate-resistant acidic phosphotase (TRAP) staining and capable of bone resorption. Addition of osteoprotegerin (OPG) to PBMC cultures does not abrogate osteoclast formation induced by LTB4. Osteoclastogenesis induced by LTB4 were dose-dependently increased but weaker than that of RANKL. Conclusion LTB4, elevated in many inflammatory diseases, is capable of inducing osteoclast formation directly by a RANKL-independent mechanism.