蛇床子素对体外培养骨髓基质干细胞增殖与成骨性分化的影响

Effects of osthol on bone marrow stromal stem cell differentiation and proliferation in vitro

目的研究蛇床子素对体外培养大鼠骨髓基质干细胞(bone marrow stromal stem cells,BMSCs)增殖与成骨性分化的影响。方法取大鼠股骨及胫骨全骨髓,利用全骨髓培养法培养单核层细胞,培养于含10%FBS的DMEM-F12培养液中,3d后首次换液,9d后传代培养。培养基中蛇床子素终浓度分别为1×10-4、1×10-5、1×10-6、1×10-7mol·L-1。增殖分析采用MTT法,于成骨性诱导培养d4、8、12、16测碱性磷酸酶(alkaline phosphatase,ALP)活性、钙盐沉积量、骨钙素分泌量,d15进行钙化结节组织化学染色及计数。成骨性诱导后不同时间点提取Total RNA,RTReal-Time PCR法检测bFGF、IGF-1、Osterix与Runx-2的基因表达情况。结果蛇床子素剂量依赖性抑制BMSCs增殖,但能明显促进其向成骨性分化,表现为提高BMSCs的ALP活性、促进骨钙素分泌、钙盐沉积量、增加钙化结节数量、提高bFGF、IGF-1、Osterix和Runx-2的mRNA表达水平。结论终浓度为1×10-5mol·L-1蛇床子素能明显促进BMSCs的成骨性分化,证明蛇床子素是中药蛇床子抗骨质疏松的有效成分。

Aim To investigate the effects of osthol on bone marrow stromal stem cells in vitro under the con-ditions of the ability to differentiate into osteoblasts and the case of proliferation. Methods The rat bone marrow sample was obtained,and the all bone marrow cell culture methods were used to separate and collect the stratum of mononuclear cells. The cells were cultured in DMEM containing 10% fetal bovine serum. Three days later the culture medium was changed for the first time. Nine days later,serial subcultivation proceeded. The final concentration of osthol was 1 × 10 -4,1 × 10 -5,1 × 10 -6,1 × 10 -7 mol·L -1 respectively. MTT method was adopted in proliferation analysis. Under the induced condition,the Alkaline phosphatase activity, calcium salt sediment yield and osteocalcin were meas-ured on 4th,8th,12th,16th day. On the fifteenth day,his-tochemistry dyeing proceeded for calcified tubercle. Total RNA was isolated and the gene expression of bFGF, IGF-1,Osterix and Runx-2 was investigated by RT-Real Time PCR. Results The BMSCs proliferation was refrained by osthol dose-dependently. But it evidently led to osteogenesis. The ALP activity,calcium salt sediment yield and osteocalcin were raised,and calcified tubercle amount was increased. Besides,it also could enhance the mRNA level of bFGF,IGF-1,Osterix and Runx-2. Conclusion The osthol with final concentra-tion of 1 × 10 -5 mol·L -1 can markedly promote BM-SCs differentiation to osteogenesis. which proves osthol is an active constituent of the traditional Chinese medi-cine Common Cnidium Fruit.