摘要
目的观察DKK1在淫羊藿总黄酮调控去势雌性大鼠骨髓间充质干细胞成骨和成脂分化平衡过程中的动态表达,为进一步阐明淫羊藿总黄酮治疗绝经后骨质疏松症的作用机制提供实验依据。方法体外分离培养去势雌性大鼠来源骨髓间充质干细胞,分别在成骨诱导液和脂肪诱导液条件下连续培养15 d,并在此基础上添加剂量为10μg/mL的淫羊藿总黄酮。采用ALP染色、ALP活性测定、油红O染色以及荧光定量PCR技术,观察淫羊藿总黄酮对骨髓间充质干细胞成骨和成脂分化的影响。用酶联免疫法(ELISA)检测淫羊藿总黄酮处理过程中DKK1蛋白的动态表达,观察DKK1蛋白在淫羊藿总黄酮调控去势雌性大鼠骨髓间充质干细胞成骨和成脂分化平衡过程中的作用。结果淫羊藿总黄酮能显著增加骨髓间充质干细胞ALP的表达以及成骨早期分化因子Runx2 mRNA的表达,显著下调骨髓间充质干细胞中脂肪形成关键基因PPARγ-2mRNA的表达,从而抑制脂滴的形成。在成骨诱导条件下,EFs呈时间依赖性下调DKK1的表达;在脂肪诱导条件下,EFs呈时间依赖性抑制DKK1蛋白的上调。结论通过抑制DKK1蛋白的表达调控去势雌性大鼠BMSCs成骨和成脂分化平衡,可能是淫羊藿总黄酮治疗绝经后骨质疏松症的分子机制之一。
Objective To observe the dynamic expression of DKKI protein in the process that epimedium-derived flavonoids （EFs） regulated the balance between osteogenic differentiation and adipogenic differentiation in bone marrow stromal cells of ovariectomized rats, and to provide an experimental evidence for the mechanism of EFs on treating postmenopansal osteoporosis. Methods Bone marrow stromal cells from ovariectomized rats were separated and cultivated in the condition of osteoinductive mediumin or liquid mediumin for 15 days in vitro. EFs （10μg/mL dose） were applied correspondingly. ALP staining, ALP activity determination, oil red 0 staining and fluorescence quantitative PCR were used to determine the influence of EFs on osteogenic differentiation and adipogenic differentiation in bone marrow stromal cells of ovariectomized rats. Moreover, in order to explore the exact mechanism of EFs on osteogenic differentiation and adipogenic differentiation in bone marrow stromal cells of ovariectomized rats, ELISA was used to determine the dynamic expression of DKK1 protein in the process. Thus, the function of DKK1 protein on regulating osteogenic differentiation and adipogenic differentiation in bone marrow stromal cells of ovariectomized rats had also been observed. Results EFs increased activity of ALP and mRNA expression of Runx2 （ early osteoblast differentiation factor） and decreased mRNA expression of PPARγ-2 （ fat generation key factor）. Importantly, EFs down-regulated expression of DKK1 protein in the osteogenic induction medium and inhibited up-regulation of DKK1 protein in the adipogenic induction medium in a time-dependent manner. Conclusion EFs regulated the balance between osteogenic differentiation and adipogenic differentiation in bone marrow stromal ceils of ovariectomized rats by down-regulating expression of DKK1 protein, which may be an important molecular mechanism of EFs on treating postmenopausal osteoporosis.
出处
《中华骨质疏松和骨矿盐疾病杂志》
2010年第4期276-284,共9页
Chinese Journal Of Osteoporosis And Bone Mineral Research
