淫羊藿对小鼠成骨细胞增殖、功能及凋亡的影响

Effects of Epimedium on proliferation, function and apoptosis of mouse osteoblasts in vitro

目的:观察淫羊藿提取液对体外培养的小鼠成骨细胞增殖、功能及凋亡的影响。方法:利用序列酶消化法从新生小鼠颅盖骨中分离和培养成骨细胞,用组织化学和免疫组化染色进行细胞鉴定。用四甲基偶氮唑盐比色法(MTT)检测淫羊藿对成骨细胞增殖的影响,通过检测成骨细胞碱性磷酸酶的活性反映淫羊藿对细胞功能的作用。建立地塞米松诱导的成骨细胞凋亡体系,并利用流式细胞仪观察淫羊藿对这一凋亡体系的影响。结果:分离和获得的大量单一多角形细胞,经碱性磷酸酶组织化学染色呈强阳性,经Ⅰ型胶原、骨桥蛋白、骨涎蛋白免疫组化染色呈阳性,证实获得的细胞为成骨细胞。淫羊藿提取液不影响成骨细胞的增殖,但可以显著提高成骨细胞碱性磷酸酶的活性,其中相当于生药1g/L组的作用最明显。100nmol/L和1000nmol/L地塞米松可以显著增加成骨细胞凋亡率,但同时加入相当于生药1g/L的淫羊藿提取液,并不引起细胞凋亡率的明显变化。结论:淫羊藿对成骨细胞的增殖和凋亡没有明显作用,但却显著增加了成骨细胞碱性磷酸酶的活性。淫羊藿防治骨质疏松症的重要机制之一可能是促进成骨细胞的成骨功能。

Objective： To investigate the effects of Epirnedium on proliferation, function and apoptosis of mouse osteoblasts in vitro. Methods： Primary osteoblasts were obtained by sequential digestion of mouse calvaria with collagenase and hyaluronidase. The identification of derived cells was done by histochemical staining of alkaline phosphatase （ALPase） and immunohistochemical staining of type I collagen, bone sialoprotein and osteopontin. MTT assay was employed to examine the proliferation of osteoblasts after treatment with Epimedium. The alkaline phosphatase activity level of mouse osteoblasts was also determined through an enzyme dynamical method. Apoptosis of osteoblasts was induced by dexamethasone and flow cytometry was utilized to examine the effects of Epimedium on the dexamethasone-induced apoptosis of osteoblasts. Results： Five populations of bone cells were obtained by sequential digestion. Osteoblasts were purely obtained by discarding the first two populations and identified by the positive staining of ALPase, type I collagen, bone sialoprotein, and osteopontin. The alkaline phosphatase activity level of osteoblasts was significantly increased by the addition of Epimedium at 0.1 -10 g/L, with the most significant increase at 1 g/L. On the other hand, the proliferation of osteoblasts was not affected after different doses of Epimedium added into the culture medium. Determined by flow cytometry, apoptosis of osteoblasts were induced by treatment with dexamethasone for 72 h. However, simultaneous administration of 1 g/L Epimedium had no effects on dexamethasone-induced apoptosis in osteoblasts. Conclusion： Epirnedium did not affect the cell proliferation and cell survival of mouse osteoblasts, but could significantly increase alkaline phosphatase activity of the cells. The increase of alkaline phosphatase activity by Epimedium in osteoblasts may be one of the important mechanisms by which Epimedium can effectively prevent osteoporosis.