大鼠长骨成骨细胞体外培养形态观察

Morphology of in vitro cultured osteoblasts derived from growing-rat long bone

目的 建立生长期大鼠长骨成骨细胞体外培养实验模型并观察其生长及骨化过程。方法 以 10周龄Wistar大鼠股骨、胫骨为材料 ,采用酶分次消化法分离成骨细胞 ,经DME :F 12加 10 %胎牛血清的培养基原代和传代培养或含 5 0 μg/mlL 抗坏血酸和 10mmol/Lβ 甘油磷酸钠的培养基连续培养 ,追踪观察成骨细胞在体外培养中的增殖及形态变化 ,并通过Giemsa、ALP及vonKossa染色技术 ,观察细胞体外基质分泌和骨化过程。结果 ①大鼠长骨成骨细胞具有体内成骨细胞的形态特征 ,增殖代谢旺盛 ,其群体倍增时间为 78h。②成骨细胞分泌形成球形和无定形基质。③当给予钙化条件培养时 ,细胞形成钙化骨基质。④大多数细胞ALP染色呈阳性并与基质的钙化密切相关。结论 培养的生长期大鼠长骨成骨细胞具有成骨细胞的某些生物学行为 ,为儿童骨生长及调控的研究提供了一种实验模型。

Objective To establish a culture system for osteoblasts from long bone of growing rats and investigate its growth and ossification process in vitro . Methods The tibiae and femora from 10-week-old Wistar rats were isolated and subjected to sequential digestions for cell isolation. Harvested cells were cultured and passaged in a DME∶F-12 1∶1 medium supplemented with 10% fetal calf serum or in medium with 50 μg/ml ascorbic acid plus 10 mmol/L beta-glycerophosphate. Cell morphology, proliferation and calcification process was observed. Results ①Cells isolated from the rat long bone showed the general morphology of osteoblasts and had population doubling time of 78 h. ② The cells synthesized spherical-like and non-shaped matrices. ③ When the cells were cultured in calcification medium and allow to grow over confluence for 3 weeks, they formed calcified nodules. ④ Most of the cells demonstrated alkaline phosphatase positive, which closely related to matrix calcification. Conclusion Osteoblasts derived from growing rat long bone by enzyme digestion exhibit some biological behaviors of osteoblasts. This simple isolation method provides a valuable model for further study of children's bone growth and regulation.