异欧前胡素对大鼠成骨细胞增殖与分化的影响

Effects of isoimperatorin on proliferation and differentiation of osteoblasts in rats

背景:植物雌激素有防治绝经后妇女骨质疏松的作用已得到公认,其作用机制之一是促进了成骨细胞的增殖与分化。目的:观察香豆素类植物雌激素异欧前胡素体外对大鼠成骨细胞增殖与分化的影响。设计、时间及地点:对比观察实验,于2006-09/2007-12在河北医科大学药学院完成。材料:出生24h内SD大鼠,异欧前胡素为中国药品生物制品检定所产品。方法:采用改良组织块法分离培养新生大鼠颅骨成骨细胞,将异欧前胡素以1×10-5～1×10-9mol/L浓度加入细胞培养体系,以未加入药物为空白对照组。作用24,48,72h后,以MTT法检测成骨细胞的增殖情况,以对硝基苯二钠基质动力学法测定细胞内碱性磷酸酶的活性,以改良Lowry法测总蛋白水平。主要观察指标:成骨细胞的增殖率和碱性磷酸酶活性。结果:大鼠成骨细胞在不同浓度异欧前胡素中培养24h,未观察到促增殖作用。培养48h后,开始出现促增殖作用增强,有效浓度为1×10-9～1×10-8mol/L。培养72h后,继续有促增殖作用,有效浓度为1×10-8～1×10-7mol/L。大鼠成骨细胞在不同浓度异欧前胡素存在下培养48h,未观察到促分化作用,培养72h后,开始出现促分化作用,有效浓度为1×10-6～1×10-5mol/L,其他浓度作用不明显。结论:异欧前胡素体外能促进大鼠成骨细胞的增殖与分化。

BACKGROUND： It is recognized that phytoestrogen has the effect of preventing post-menopausal osteoporosis and one of the mechenisms undering it is that it can stimulate the proliferation and differentiation of osteoblasts. OBJECTIVE： To study the effects of isoimperatorin （coumarin phytoestrogen） on the proliferation and differentiation of osteoblasts in vitro. DESIGN, TIME AND SETTING： A controlled observational experiment was performed at the Faculty of Pharmacy, Hebei Medical University from September 2006 to October 2007. MATERIALS： New born SD rats were used. Isoimperatorin was provided by Medicine Inspection Institute of China. METHODS： Osteoblasts isolated from neonatal rat calvaria were cultured by modified tissue culture technique. Then some of them were cultured in vitro with isoimperatorin of 1 ×10^- 5 mol/L to 1 ×10^-9 mol/L, and others were used as the blank control. At hour 24, 48 and 72 following culture, MTT and p-nitrophenylphosphate （PNPP） were used to observe separately the proliferation and the activity of alkaline phosphatase （ALP） of osteoblasts cultured. Total protein was measured with modified Lowry method. MAIN OUTCOME MEASURES： The proliferation rate and the ALP activity of osteoblasts. RESULTS： It was not uniti 148 hours of culture that isoimperatorin began to show its effects of promoting proliferation of rat osteoblasts, and the effective concentrations ranged from 1 ×10^- 9 mol/L to 1 ×10^-8 mol/L; as for culture of 72 hours, the effective concentrations was between 1×10^-8 mol/L and 1×10^-7 mol/L. In addition, the differentiation of osteoblasts was stimulated by isoimperatorin only after 72 hours of culture, and only the concentration between 1 ×10^- 6 mol/L and 1 ×10^- 5 mol/L was effective obviously. CONCLUSION： Isoimperatorin can stimulate the proliferation and differentiation of cultured osteoblasts in vitro.