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目的探讨骨髓间充质干细胞经成骨细胞诱导不同时间后的细胞性质。方法成人骨髓分离单个核细胞(MNC),体外培养获得间充质干细胞(MSC),传至第4代后加入地塞米松(DEX)、抗坏血酸和β-甘油磷酸,诱导成骨分化。诱导7d和14d后的细胞与正常成人成骨细胞分别加入不同浓度骨形态发生蛋白-2(BMP-2)和DEX,48h后检测细胞增殖、碱性磷酸酶(ALP)活性和骨钙素(OCN)的含量。结果BMP-2和DEX对诱导7d后的细胞有明显的刺激增殖和ALP活性的作用;对诱导14d后的细胞,BMP-2和DEX都不能刺激细胞增殖和ALP活性,但OCN含量明显增加。正常成人成骨细胞对BMP-2和DEX作用的反应与诱导14d的成骨样细胞基本一致,但其OCN绝对含量明显高于MSC来源的成骨细胞。结论骨髓MSC向成骨诱导7d和14d后细胞分化程度不同。诱导时间在MSC的成骨分化中是一个非常关键的因素,但其机制和最佳诱导时间尚需进一步研究。诱导14d后的MSC的成骨活性低于正常成人成骨细胞。
Objective To investigate the cellular properties of bone marrow mesenchymal stem cells (BMSCs) induced by osteoblasts at different time points. Methods MNCs were isolated from adult bone marrow and cultured in vitro. Mesenchymal stem cells (MSCs) were obtained in vitro. After passage 4, dexamethasone (DEX), ascorbic acid and β-glycerophosphate were added to induce osteogenic differentiation. After induced for 7d and 14d, the cells were incubated with normal adult osteoblasts for different concentrations of bone morphogenetic protein-2 (BMP-2) and DEX for 48h. The cell proliferation, alkaline phosphatase (ALP) activity and osteocalcin OCN) content. Results Both BMP-2 and DEX significantly stimulated proliferation and ALP activity of cells 7 days after induction. Both BMP-2 and DEX did not stimulate cell proliferation and ALP activity after 14 days, but the content of OCN increased significantly. The response of normal adult osteoblasts to BMP-2 and DEX was basically the same as that of osteoblast-like cells induced for 14 days, but the absolute content of OCN was significantly higher than that of MSC-derived osteoblasts. Conclusion The differentiation of bone marrow MSCs after 7d and 14d osteogenic differentiation is different. Induction time in osteogenic differentiation of MSC is a very crucial factor, but the mechanism and the optimal induction time needs further study. The osteogenic activity of MSC after 14 days of induction was lower than that of normal adult osteoblasts.