诱导法与机械法体外培养破骨细胞的比较研究

目的 探讨不同方法培养的破骨细胞(OC)在形态学及骨吸收功能的差异,为体外培养OC提供方法学实验依据.方法 采用机械分离的方法,即从1d龄SD大鼠四肢长骨机械分离获得成熟OC；诱导法,即利用RANKL(100 ng/mL)和M-CSF(100 ng/mL)诱导RAW264.7细胞形成破骨样细胞(OLC).对获得的OC和OLC进行形态学和骨吸收功能观察比较.结果 OC和OLC均为TRAP染色阳性的多核巨细胞,与细胞共培养骨片上可形成骨吸收陷窝;与机械分离法比较,诱导法培养出的OLC数目较多,但骨吸收陷窝较小而浅.结论 直接分离培养法可获得骨吸收功能较活跃的OC,但数目较少,适合骨吸收功能分析、破骨迁移黏附、凋亡研究及单细胞分子生物学研究.诱导形成的OLC数量较多,但骨吸收功能较差,适合用于OC分化发育过程的研究.

Comparison of the Culturing Methods of Osteoclasts between Induced and Mechanical Procedure

Objective To investigate the differences of morphology and bone absorption function of osteoclast(OC)between different cultural methods,and to provide the basis for further investigation on the OC in vitro.Methods Mechanical separation method was used to isolate mature osteoclasts from long bones of SD rats aged one-day,and induced culture method was applied to induce osteoclast-like cell(OLC) by using RANKL(100 ng/mL)and M-CSF(100 ng/mL).The morphological and functional changes of OC as well as OLC were observed.Results OC and OLC were multinucleated giant cells,which could be stained positively by TRAP,and could form bone absorptive lacuna.The number of OLC in inducement method was more than in mechanical separation method,but bone absorption lacuna in mechanical separation method was smaller and shallower as compared with inducement.Conclusions OC derived directly from long bones is less in number,but is more active in bone absorption,thus can be appropriate for the study of bone absorption,migration and adhesion,apoptosis and monocellular molecular biology.OLC obtained by inducement method is more in number,but less active in bone resorption and is better for the study of OC differentiation and development.