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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 rat model of long osteoblasts in vitro and to observe its growth and ossification process. Methods Osteoblasts were isolated from the femur and tibia of 10-week-old Wistar rats by enzymatic fractionated digestion. Primary and subculture with DME: F 12 plus 10% fetal bovine serum or medium containing 50 μg / ml L ascorbic acid and 10 mmol / L sodium glycerophosphate. The proliferation and morphological changes of osteoblasts in vitro were observed. The secretion and ossification of the cells in vitro were observed by Giemsa, ALP and von Kossa staining. Results ① The long osteoblasts in rat had the morphological characteristics of osteoblasts in vivo and proliferative metabolism was strong. The population doubling time was 78h. ② osteoblasts secreted to form a spherical and amorphous matrix. ③ When given calcification conditions, the cells form calcified bone matrix. Most cells ALP staining was positive and closely related to matrix calcification. Conclusion The long osteoblasts cultured in the growing stage have certain biological behaviors of osteoblasts and provide an experimental model for the study of bone growth and regulation in children.