淫羊藿苷对体外培养乳鼠颅骨成骨细胞增殖、分化及成熟的影响

Effects of Icariin on the Proliferation,Differentiation and Maturation of Rat Calvarial Osteoblasts in vitro

目的:研究淫羊藿苷对体外培养大鼠乳鼠颅骨成骨细胞(Rat calvarial osteoblasts,ROB)增殖和分化成熟的影响。方法:取新生SD大鼠颅骨,多次酶消化法培养ROB,每3 d换液1次,铺满80%皿底后传代培养。采用终浓度分别为1×10-4、1×10-5、1×10-6、1×10-7mol/L的淫羊藿苷进行药物干预,MTT法检测细胞增殖率,同时检测成骨性诱导培养9 d后的碱性磷酸酶(Alkaline phosphatase,ALP)活性,筛选出最佳浓度。以最佳药物浓度干预ROB的分化成熟,比较淫羊藿苷组和不加药的对照组之间的钙化结节数量及碱性磷酸酶阳性克隆数(coloniesstained positive for alkaline phosphatase,CFU-FALP);提取总RNA,Real Time RT-PCR检测Runx-2与Osterix mRNA的表达情况;Western-blot法检测Ⅰ型胶原蛋白的分泌量。结果:淫羊藿苷剂量依赖性抑制细胞的增殖,但可强烈促进ROB的分化成熟,最佳药物浓度为1×10-5mol/L。该浓度下的淫羊藿苷可显著增加钙化结节和CFU-FALP数量,促进与成骨相关的转录因子Runx-2与Osterix mRNA的表达,同时也可增强Ⅰ型胶原蛋白的分泌。结论:浓度为1×10-5mol/L的淫羊藿苷可显著促进ROB的分化成熟,提示淫羊藿苷有较强的促骨形成活性,具备开发为抗骨质疏松新药的潜力。

Objective：To investigate the effects of icariin on the proliferation,differentiation and maturation of rat calvarial osteoblasts（ROB）.Methods：Segregated neonatal SD rat skull,enzyme digestion to obtain ROB.The culture medium was replaced every three days.Serial subcultivation proceeded when cells covered with 80% culture dish.Icariin was added into the culture at 1×10-4、1×10-5、1×10-6、1×10-7 mol/L respectively.MTT method was adopted in proliferation analysis.The activity of ALP was assayed after 9 days′ induced.Optimal concentration icariin was added into the medium,then the osteogenic differentiation markers including mineralized bone nodules,CFU-FALP were compared between the icariin-added group and the control.Total RNA was isolated and the gene expressions of Runx-2 and Osterix were investigated by Real Time RT-PCR.Total protein was also isolated and the secretion of collagenⅠwas examined by Western-blot.Results：The ROB proliferation was inhibited by icariin in a dose-dependent manner.But it evidently led to osteogenic process and maturation.1×10-5 mol/L was the best concentration.Icariin improved the secretion of collagenⅠ,CFU-FALP amounts and mineralized nodules significantly.It also enhanced the mRNA level of Runx-2 and Osterix.Conclusion：The icariin with final concentration of 1×10-5 mol/L can enhance the osteogenic differentiation and maturation of ROB significantly,suggesting that icariin has the activity of inducing bone formation,it has the potential to be developed into a new drug of anti-osteoporosis.