目的:观察辛伐他汀(SIM)对体外人骨髓基质干细胞(hBMSCs)向成骨分化的影响,探讨其刺激成骨的作用机制.方法:取健康成人骨髓进行体外成骨诱导培养,实验分为对照组(G1):不给予药物干预;实验组(G2):传代后加入SIM1×10~(-7)mol/L.于传代后7,14,21 d采用Real time RT-PCR检测mRNA水平和BMP-2,Smadl,B-catenin,Frizzled2的表达,并用免疫组织化学检测蛋白水平p-catenin的表达.传代后7 d行细胞碱性磷酸酶(ALP)染色和比活性检测;传代后21 d行yon Kossa染色,检测细胞外基质矿化.结果:SIM作用后7,14,21 d 3个不同时间点,BMP-2表达均显著高于对照组;Smadl在成骨诱导14,21 d时表达高于G1,其余组间差异均不显著;β-catenin免疫组织化学染色2组差异不显著;G2组ALP表达和矿化能力均显著高于G1组.结论:SIM 1×10~(-7)mol/L可促进体外成骨诱导培养的hBMSCs向成骨细胞分化,上调TGF-β/BMPs信号通路部分信号分子表达,但未能显著改变Wnt/β-catenin信号通路部分相关因子的表达.
Effects of simvastatin on differentiation of osteoblasts derived from human bone marrow stromal cells
AIM: To investigate the effects of simvastatin (SIM) on differentiation of osteoblast derived from the cultured human bone marrow stromal cells ( hBMSCs ). METHODS: hBMSCs derived from human were cultured in vitro and divided into 2 groups: Control group(Gl ) ,SIM group(G2). After subculturing, the medium of treatment group (G2) were added 10~(-7) M simvasta-tin. Real time RT-PCR was performed to evaluate the mRNA expressions of BMP-2, Smadl, β-catenin, LRP5, Frizzled2 at day 7,14 and 21 after subculturing; The protein level expression of β catenin was assessed by immuhistochemistry staining; Alkaline phosphatase staining was used to assess osteoblast differentiation at day 7 after subculturing; Von Kossa staining was used to assess extracellular matrix mineralization and at day 21 after subcultu ring. RESULTS: The expression levels of mRNA of BMP2 in group G2 were significantly higher than those of group Gl at 3 time points; The expression levels of mRNA of Smad1 in group G2 were significantly higher than those of group Gl at 14 and 21 d subculturing, and the mRNA expression levels of other detected genes showed no significant difference between two groups at each time point. Immuhistochemistry staining; No significant difference was found of the protein expression levels of β-catenin at the 3 time points between the 2 groups, the expression level of ALP and the capability of extracellular matrix mineralization in G2 group was significantly higher than that of Gl group. CONCLUSION: Treat ment with simvastatin( 1 x 10~(-7) mol/L) could promote osteoblasto genesis of HBMSCs in vitro, probably partially from activing TGF-β/BMPs signaling pathway and upregulating expression of its signaling moleculars. No change was found of expression levels of the detected genes of Wnt/β-catenin signaling pathway after simvasta-tin treatment, further study should be performed to detect the role of Wnt/β-catenin signaling pathway in osteoblastogenesis-promo-ting effect of simvastatin on hBMSCs.
simvastatinhuman bone marrow stromal cellsosteoblastBMP-2real time RT-PCR
NETL
NSTL
万方数据
维普
