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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。 【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
【Objective】 To establish a rat model of long osteoblasts cultured in vitro. 【Methods】 Osteoblasts were isolated from the femur and tibia of 10-week-old Wistar rats by enzymatic fractionated digestion. The primary and subculture cultures of DME: F 12 plus 10% fetal bovine serum were used to observe the effects of The morphological changes of osteocytes were observed in vitro. The cells were identified by alkaline phosphatase (ALP), Van Gieson’s collagen fibers and von Kossa staining. 【Results】 (1) The cultured cells had morphological characteristics of osteoblasts. Ex vivo proliferative metabolism was strong, and their population doubling time was 78 hours. ② cells with high alkaline phosphatase activity, with the passage of a slight increase. ③ cultured cells can synthesize secreted collagen fibers. ④ After three weeks of culture under calcification conditions (50 μg / ml ascorbic acid, 10 mM β-glycerophosphate), cells formed calcified bone matrix in vitro. 【Conclusion】 The long osteoblasts in the growing stage of rat have obvious biological characteristics of osteoblasts and provide an objective experimental method for further studying the mechanism of bone growth and metabolism