一氧化氮对小鼠原代成骨细胞的增殖及分化调控

Regulation of nitric oxide in the proliferation and differentiation of mouse primary osteoblast-like cells

目的:探讨一氧化氮(nitric oxide,NO)对小鼠原代成骨细胞的矿化调控作用。方法:分离培养小鼠原代成骨细胞,给予外源性NO供体S-亚硝基N-乙酰基青霉胺(SNAP)2周,检测成骨细胞增殖活性和碱性磷酸酶活性。Von Kossa方法检测成骨细胞矿化结节形成情况,免疫组化方法检测骨唾液酸蛋白(BSP)表达。结果:NO对成骨细胞增殖具有双向作用:在β-甘油磷酸钠加抗坏血酸存在下,72h内在一个比较宽的浓度范围(10-2μmol/L～101μmol/L)NO供体SNAP呈剂量依赖性地刺激成骨细胞样细胞生长,高于102μmol/LSNAP则产生抑制作用。和β-甘油磷酸钠加Ascorbic acid作用组相比,SNAP可明显增加碱性磷酸酶活性,Von Kossa检测可观察到细胞间钙盐沉积,两组药物都能促使成骨样细胞骨唾液酸蛋白表达,而SNAP单独应用无明显矿化作用。结论:适量浓度一氧化氮,以β-甘油磷酸钠和抗坏血酸为介质,可加速小鼠原代成骨细胞增殖分化,并可能通过调控骨唾液酸蛋白表达刺激骨钙化。

To explore the effect of nitric oxide on the biomineralization in mouse primary osteoblast-like cells. Method： Mouse primary osteoblasts were isolated and cultured.Cell proliferation assay, alkaline phosphatase （ALP）ac-tivity and mineralization were detected in different groups with the treatment of S-nitroso-N-acetyl-dl-penicillamine （SNAP） alone, β-glycerophosphate sodium and ascorbic acid,SNAP plus β-glycerophosphate sodium and ascorbic acid （SNAP plus group）.The expression of bone sialoprotein （BSP）was examined by immunohistochemistry. Result： NO may play biphasic effect on osteoblasts. SNAP significantly increased osteoblast＇s proliferation activity with the existence of β-glyc-erophosphate sodium and ascorbic acid in a dose-dependent manner between the concentration of 10^-2μmol/L～10^1μmol / L.Compared with the effect of β-glycerophosphate sodium and ascorbic acid, SNAP plus group promoted the ALP activity of osteoblasts, and the expression of BSP was also detected in these two groups from the 12 th day of culture, while SNAP alone didn＇t function.Conclusion： NO may stimulate the proliferation and differentiation of mouse primary osteoblast-like cells through regulating the expression of BSP, therefore promote bone calcification.