摘要
目的:比较分析破骨细胞(osteoclast,OC)体外分离培养的方法,并动态观察其形态、分化及功能,为进一步探讨药物对OC性骨吸收的作用奠定基础。方法:采用两种OC的培养方法,即从新生24h的兔长干骨骨髓腔内壁机械分离成熟OC和1,25(OH)2维生素D3诱导鼠骨髓细胞形成破骨样细胞(osteoclastlikecell,OLC)的方法对获得的OC进行形态学和功能观察。结果:倒置显微镜下观察,OC是一种多核巨细胞,有伪足并借助伪足的凹凸伸缩而变化形态、运动行走;HE染色均可见OC多核、胞浆有空泡;特异性抗酒石酸酸性磷酸酶(tartrateresistanacidphosphatase,TRAP)染色可见OC胞浆阳性,呈酒红色,多核。体外OC与骨片共同培养形成骨吸收陷窝,且在培养的7d内陷窝数目和面积逐渐增大。1,25(OH)2维生素D3诱导鼠骨髓细胞形成OLC,其形态结构特征与成熟OC相同,诱导出的破骨样细胞数目多于机械分离的方法,但每个细胞胞核数目总体上少于机械分离的方法,而且胞核多位于细胞周边,在骨片上形成的陷窝也较小而浅;培养液上清中的钙离子含量和酸性磷酸酶(acidphosphatase,ACP)含量在培养的1～12d内随时间逐渐增加。结论:针对不同实验目的可以采用不同的分离培养OC方法。从骨髓腔内壁机械分离培养的方法可获得骨吸收功能较活跃的OC。1,25(OH)2维生素D3诱导鼠骨髓细胞形成的OLC数量较多,适于对OC分化过程的研究。
Objective ： To study the origin, morphological structure, and functional regulation of osteoclast（OC） for further investigation on the mechanism and regulation of bone resorption. Methods： The OCs were isolated by two kinds of traditional method. Osteoclasts were isoclated from neonatal rat long bones. The cytochemistry was observed. The osteoclast-like cells （OLC） were derived from the mouse bone marrow cells in the presence of 1,25 （OH）2VitD3 in vitro. Results： Both morphological and functional studies showed that the isolated cells shared some of the typical characteristics of osteoclasts, that is A. muhinuclearity; B. developing spreading and pseudopodial activity when cultured on glass; C. high tartrate-resistant acid phosphatase （TRAP） ; D. resorption lacunae could be found when the cells were cocultured with devitalized bone slices and the number was increased as the time followed. OLC had the same histological and structural traits as the OCs by the former method. The concentration of Ca^2＋ and acid phosphatase （ACP） increased gradually. Conclusion： Different kinds of method fit different experiments. The OC obtained by the first method has more activity of bone resorption. The OLC by the second method has more in quantity and can be used in the study of cell differentiation.
出处
《北京大学学报（医学版）》
CAS
CSCD
北大核心
2005年第5期536-541,共6页
Journal of Peking University:Health Sciences
基金
国家自然科学资金项目(30271412)
北京大学211工程口腔医学学科群资助(KQ0221116)~~
