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目的:建立在体外培养时能保持分化表型的成骨细胞实验模型,并观察成骨细胞在体外的凋亡.方法:用酶解的方法从新生SD大鼠颅骨分离成骨细胞;用改良钙钴法及偶氮染色法测定其碱性磷酸酶(AKP)活性;用Von Kossa染色法及Fluo-3染色法检测其钙化沉积物;根据凋亡细胞中乙酰胆碱酯酶(AChE)高表达的特性,用乙酰胆碱酯酶染色法观察不同时期的凋亡细胞.结果:原代大鼠颅骨成骨细胞在体外培养44天中,逐步形成包括多层细胞结节,钙化的细胞外基质组成的骨样组织,随着基质的钙化,凋亡细胞逐步增多.结论:大鼠颅骨成骨细胞在体外发育过程的表型能反映成骨细胞在体外的成熟过程,是研究成骨细胞生物学理想的实验模型.
OBJECTIVE: To establish an experimental model of osteoblast that can maintain the differentiated phenotype in vitro and to observe the in vitro apoptosis of osteoblasts.Methods: Osteoblasts were isolated from the skull of newborn SD rats by enzymolysis method, The alkaline phosphatase (AKP) activity was detected by calcium-cobalt method and azo-dye method. The calcified sediment was detected by Von Kossa staining and Fluo-3 staining. According to the high expression of acetylcholinesterase (AChE) in apoptotic cells And the apoptotic cells were observed by the method of acetylcholinesterase staining.Results: The primary rat calvarial osteoblasts were gradually cultured for 44 days in vitro, including the formation of multilamellar cell nodules and calcified extracellular matrix Like tissue, with the matrix calcification, apoptotic cells gradually increased.Conclusion: The phenotype of rat calvarial osteoblasts during in vitro development can reflect the maturation process of osteoblasts in vitro and is ideal for studying the biology of osteoblasts Experimental model.