淫羊藿苷促进羊骨髓间充质干细胞的增殖和成骨分化

Icariin enhances proliferation and osteogenic differentiation of goat bone marrow mesenchymal stem cells

背景:寻找一种简单有效、安全价廉且可替代生长因子的生物活性物质,是骨组织工程的客观要求,中药来源的植物黄酮淫羊藿苷可通过提高成骨细胞功能促进成骨,但是对骨髓间充质干细胞的作用尚未明确。目的:探讨淫羊藿苷对羊骨髓间充质干细胞增殖和分化的影响。设计、时间及地点:细胞学体外观察,于2008-09/2009-02在南方医院创伤骨科实验室完成。材料:清洁级成年雄性中国青山羊3只,由南方医院实验动物中心提供。淫羊藿苷标准品由中国药品生物制品检定所提供,批号110737-200312,纯度98.3%。方法:体外分离培养羊骨髓间充质干细胞,传至第3代后,按2×103/孔接种至96孔板,分别加入100,10,1,0.1μmol/L淫羊藿苷培养基150μL进行培养;设立对照组,仅加入等量普通DMSO培养基。主要观察指标:采用MTT法检测细胞生长曲线,流式细胞仪测定细胞周期,通过碱性磷酸酶试剂盒测定细胞碱性磷酸酶活性,放免法检测细胞骨钙素表达,茜素红染色观察钙化结节形成能力。结果:0.1μmol/L淫羊藿苷可明显促进羊骨髓间充质干细胞增殖,增加S期和G2/M期细胞数量;随着淫羊藿苷浓度的增加,促细胞增殖活性降低,100μmol/L淫羊藿苷表现为明显的抑制细胞增殖作用。淫羊藿苷呈剂量依赖性促进羊骨髓间充质干细胞碱性磷酸酶活性和骨钙素表达。10,100μmol/L淫羊藿苷诱导钙化结节形成能力优于0.1和1μmol/L淫羊藿苷,对照组羊骨髓间充质干细胞未形成钙化结节。结论:淫羊藿苷对羊骨髓间充质干细胞的增殖和成骨分化具有促进作用,可被视为一种良好的骨诱导活性因子。

BACKGROUND： The objective requirement of bone tissue engineering is to seek a simple, effective, safe, cheap bioactive substance, which can substitute growth factor. Icariin, a plant-derived fiavonol glycoside, has ability of promoting bone formation by improving osteoblast activities. However, the effects of icariin on bone marrow mesenchymal stem cells （BMSCs） are unclear. OBJECTIVE： To study the effects of icariin on proliferation and osteogenic differentiation of goat BMSCs. DESIGN, TIME AND SETTING： The cytology in vitro study was performed at the Laboratory of Department of Orthopaedics and Traumatology, Nanfang Hospital from September 2008 to February 2009. MATERIALS： Three clean adult goats were supplied by Experimental Animal Center, Nanfang Hospital. Icariin standard preparation was obtained from the National Institute for the Control of Pharmaceutical and Biological Products, lot number 110737-200312, with a purity of 98.3%. METHODS： Goat BMSCs were isolated and cultured in vitro. At the third passage, goat BMSCs at 2×10^3/well were incubated in a 96-well plate, and treated with 150 μL 100, 10, 1, 0.1 μmol/L icariin medium. BMSCs in the control group were treated with an equal volume of dimethyl sulphoxide （DMSO） medium. MAIN OUTCOME MEASURES： Growth curves were examined by MTT assay. Cell cycle was analyzed by flow cytometry. Alkaline phosphatase and osteocalcin were detected by alkaline phosphatase kit and radio-immunity method, respectively. Calcified nodules were observed using alizarin red S staining. RESULTS： 0.1 μmol/L icariin improved proliferation of goat BMSCs by increasing the proportion in S and G2/M phase. With the increased concentration of icariin, the activity of promoting cells decreased. 100 μmol/L icariin showed a depressant effect on proliferation. Icariin played a dose dependence, and promoted Alkaline phosphatase activity and osteocalcin expression in goat BMSCs. Calcified nodule formation induced by 10, 100 μmol/L icariin was better compared with 0.1 and 1 μmol/L icariin. Calcified nodules were not found in goat BMSCs in the control group. CONCLUSION： Icariin enhances proliferation and osteogenic differentiation of goat BMSCs and can be used as an osteoinductive factor.