【摘要】 【目的】　建立生长期大鼠长骨成骨细胞体外培养实验模型。　【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 ,追踪观察成骨细胞在体外培养中的形态变化 ,并通过碱性磷酸酶 (ALP)、Van Gieson氏胶原纤维及vonKossa组化染色技术 ,对细胞进行鉴定。　【结果】　①培养细胞具有成骨细胞的形态特征 ,体外增殖代谢旺盛 ,其群体倍增时间为 78小时。②细胞具有较高的碱性磷酸酶活性 ,随传代而略有增加。③培养细胞能合成分泌胶原纤维。④当给予钙化条件 (5 0 μg/ml抗坏血酸 ,10mMβ 甘油磷酸钠 )培养 3周后 ,细胞在体外形成钙化骨基质。 　【结论】　培养的生长期大鼠长骨成骨细胞具有明显的成骨细胞生物学特性 ,为进一步研究骨生长代谢及调控机制提供了一种客观的实验手段更多还原

【Abstract】 Objective To establish osteoblasstsic cell isolaton and in vitro culturing method from young growing rat long bone. Methods Tibiae and femora from 10 week old Wistar rat were isolated and subjected to sequential digestions in 0.25% trypsin and 0.1%collagenase.Harvested cells were cultured in a DME:F 12 1∶1 medium supplemented with 10% fetal calf serum,in which the cell growth and proliferation were observed.In addition,the studies utilized established protocols for the characterization of these cells as osteoblasts by means of alkaline phosphatase activity,Van Gieson and von Kossa staining. Results ①Cells released from young growing rat long bone had the general morphology of osteoblasts and undergo proliferation and differentiation.The population doubling time was 78 hr.②Most of the cells were alkaline phosphatase positive.There was a little elevated positive rate after passage.③The cells retained the ability to produce and synthesize collagen.④When the cell layers were supplemented with both 50μ g/ml ascorbic acid and 10 mM beta glycerophosphate and allow to grow past confluency for 3 weeks,they formed calcified nodules. Conclusions Osteoblastic cells derived from young growing rat long bone by enzymatic digestion exhibited some biologic behaviors of osteoblasts.This simple isolation technique now facilitated the availability of young growing rat osteoblastic cells for further investigation of bone growth regulation,metabolism and biology.更多还原