金雀异黄酮促进体外培养大鼠骨髓间充质干细胞成骨性分化的机制研究

A Mechanism Study on Genistein in Promotion of Osteogenic Differentiation of Rats Marrow Mesenchyme Stromal Cell in Vitro Culture

目的探讨金雀异黄酮促进体外培养原代大鼠骨髓间充质干细胞(rats bone marrow mesenchymal stem cell,r BMSC)成骨性分化的机制。方法体外分离培养r BMSC传代3次后,采用碱性磷酸酶(ALP)活性筛选金雀异黄酮最佳浓度,采用终浓度为1×10^(-5)mol/L的金雀异黄酮对体外培养原代r BMSC进行干预;药物干预后48和72 h采用甲基噻唑基四唑(MTT)测定细胞增殖情况;在干预后3、6、9、12和15 d测定ALP活性、钙盐沉积量;干预后24、48和96 h检测OPG和RANKL mRNA表达水平。结果金雀异黄酮组处理r BMSC 48和72 h后光密度值高于对照组(P<0.05);干预rBMSC 12和15 d后金雀异黄酮组ALP活性高于对照组,干预6、9、12和15 d后钙盐沉积量高于对照组,干预24、48和72 h OPG mRNA水平高于对照组,干预72 h RANKL mRNA高于对照组,干预48 h OPG/RANKL高于对照组(P<0.05,P<0.01)。结论 1×10^(-5)mol/L金雀异黄酮可促进体外培养r BMSC成骨性分化,其作用可能是通过OPG/RANKL途径实现的。

Objective To investigate mechanism of Genistein in promotion of osteogenic differentiation of rats bone marrow mesenchymal stem cell（ rBMSC）. Methods r BMSCs were cultured in vitro for 3 times of generation,and optimal Genistein concentration was tested by alkaline phosphatase（ ALP） activity screening,and primary r BMSCs were intervened by final concentration 1 × 10^-5mol/L Genistein in vitro. After drug intervention for 48 and 72 h,conditions of cell multiplication were detected by methyl thiazolyl tetrazolium（ MTT） assay. ALP activities and calcium salt sediment yield were detected on 3^rd,6^th,9^th,12^th and 15^th d drug intervention. OPG and RANKL mRNA expressions were detected at 24^th,48^th and 72 thh drug intervention. Results Compared with those in control group,in Genistein group,optical density values were higher after 48 and 96 h of Genistein intervention（ P〈0. 05）; ALP activities were increased after 12 and 15 d of Genistein intervention; values of calcium salt sediment yield were higher after 6,9,12 and 15 d of Genistein intervention; OPG mRNA expressions were increased after 24,48 and 72 h of Genistein intervention,and RANKL mRNA expression was significantly increased after 72 h of Genistein intervention,and OPG/RANKL level was higher after48 h of Genistein intervention（ P〈0. 05,P 〈0. 01）. Conclusion 1 × 10^-5mol/L Genistein can promote osteogenic differentiation of marrow mesenchymal stem cell,and its effect may achieve through OPG/RANKL signal.