淫羊藿总黄酮对体外培养骨细胞功能的影响

Effect of total flavonoids of herba epimedii on osteoblast and osteoclast cultured in vitro

目的 :观察淫羊藿总黄酮 (HEF)对体外培养成骨细胞以及破骨细胞功能的影响。方法 :分别用酶消化法和体外机械分离法获得新生SD大鼠成骨细胞、破骨细胞 ,在培养液中分别加入不同浓度的HEF ,观察成骨细胞的增殖功能、分化功能和矿化功能。以抗酒石酸酸性磷酸酶染色观察破骨细胞数目、形态 ,Image ProPlus图像软件分析骨片上骨吸收陷窝的数目和面积。结果 :增殖率测定HEF浓度为 10 - 4mol·L- 1时与对照组比较差异有非常显著意义 (P <0 .0 1) ;碱性磷酸酶活性浓度≥ 10 - 8mol·L- 1,差异有显著意义 ;矿化结节面积/孔HEF 10 - 10mol·L- 1组和 10 - 4mol·L- 1组与对照组相比均增加(P <0 .0 1)。骨片培养 3d ,吸收陷窝计数的结果显示 ,各浓度对破骨细胞吸收功能均有抑制作用 ,仅10 - 4mol·L- 1组有统计学意义 (P <0 .0 5 )。陷窝面积 10 - 10 mol·L- 1组和 10 - 4mol·L- 1组与对照组相比 ,均无显著意义 (P >0 .0 5 )。结论 :HEF可以通过影响成骨细胞增殖、分化、矿化促进骨形成 ;而在体外减少破骨细胞数目 ,减弱破骨细胞吸收功能。

AIM: To observe the effects of total flavonoids of herba epimedii (HEF) on osteoblast and osteoclast cultured in vitro. METHODS: The osteoblasts and osteoclasts of newborn SD rats were obtained through the enzyme dissociation method and mechanical isolated method, respectively. The different HEF concentrations (10 -10-10 -4 mol·L -1) were added into the culture medium, and the proliferation, differentiation and mineralization of the osteoblast were observed. Dynamic changes in numbers, morphology of the osteoclast were detected with tartrate resistance acid phosphatase (TRAP) staining. The resorption pits and areas on slices were counted by Image-Pro Plus. RESULTS: When HEF was detected with proliferation rate at 10 -4 mol·L -1, There was significant difference compared with the control group (P <0.01). The alkaline phosphatase activity significantly increased at HEF≥10 -8 mol·L -1. The mineralized areas/well were significantly increased compared among the 10 -10, 10 -4 mol·L -1, and control groups (P< 0.01) . After 3 d incubation, the numbers of the resorption pits demonstrated that there was inhibition of resorption of osteoclasts at different concentrations. There was statistic difference only at 10 -4 mol·L -1 (P< 0.05). There were no significant differences at areas of the resorption pits among 10 -10 mol·L -1, 10 -4 mol·L -1 and the control groups (P> 0.05). CONCLUSION: HEF can not only stimulate formation of bone through affecting the proliferation, differentiation and mineralization of osteoblasts, but also decrease the numbers of the osteoclasts and inhibit osteoclast resorption activity in vitro directly.