Mechanism of osteogenic differentiation for mesenchymal stem cells induced by BMP9 and Wnt3a in vitro
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摘要:
目的：探讨经典Wnt信号通路与骨形态发生蛋白9（Bone morphogenetic protein 9，BMP9）诱导间充质干细胞（Mes－enchymal stem cells，MSCs）成骨的相互影响及分子机制。方法：以小鼠骨髓来源MSCs C3H10T1/2为目的细胞，用Wnt3a上调经典Wnt信号通路，联合BMP9作用于细胞，通过碱性磷酸酶（Alkaline phosphatase，ALP）活性测定、钙盐沉积实验、real time PCR、免疫细胞化学染色等方法观测BMP9与经典Wnt信号通路联合后对MSCs成骨分化的影响。结果：Wnt3a明显促进了BMP9诱导的ALP活性的增加[F=264.962，P=0.000；t（BMP9+Wnt3a）-BMP9=7.19，P=0.000；t（BMP9+Wnt3a）-Wnt3a=16.36，P=0.000]，同时也明显增加了成骨标志物骨钙蛋白（Osteocalcin，OC）[F=3 920.102，P=0.000；t（BMP9+Wnt3a）-BMP9=39.10，P=0.000；t（BMP9+Wnt3a）-Wnt3a=62.56，P=0.000]和骨桥蛋白（Osteopontin，OPN）[F=1 935.824，P=0.000；t（BMP9+Wnt3a）-BMP9=40.88，P=0.000；t（BMP9+Wnt3a）-Wnt3a=56.42，P=0.000]的表达以及Runx2的mR－NA的表达[F=3 635.980，P=0.000；t（BMP9+Wnt3a）-BMP9=79.37，P=0.000；t（BMP9+Wnt3a）-Wnt3a=86.96，P=0.000]，并且还促进了钙盐沉积。结论：Wnt3a与BMP9相互协同诱导MSCs的骨向分化，其中成骨转录因子Runx2可能作为二者的交汇点起了重要作用。
Abstract:
Objective：To analyze the molecular mechanism of osteogenic differentiation of mesenchymal stem cells（MSCs） induced by canonical Wnt3a signal pathway and bone morphogenetic protein 9（BMP9）. Methods：C3H10T1/2 cells were infected by recombinant adenovirus expressing BMP9 and Wnt3a，and then alkaline phosphatase（ALP），the early osteogenic marker，was detected by quantita－tive assay. Osteogenic differentiation marker at later stage，calcium deposition was determined by Alizarin Red S staining. The protein levels of osteocalcin（OC） and osteopontin（OPN） were detected by immunocytochemical staining. The expression levels of OC，OPN and Runx2 were analyzed by real time PCR. Results：Wnt3a enhanced the ability of BMP9-induced ALP activity（F=264.962，P=0.000；t（BMP9+Wnt3a）-BMP9=7.19，P=0.000；t（BMP9+Wnt3a）-Wnt3a=16.36，P=0.000） and increased expressions of OC（F=3 920.102，P=0.000；t（BMP9+Wnt3a）-BMP9=39.10，P=0.000；t（BMP9+Wnt3a）-Wnt3a=62.56，P=0.000） and OPN（F=1 935.824，P=0.000；t（BMP9+Wnt3a）-BMP9=40.88，P=0.000；t（BMP9+Wnt3a）-Wnt3a=56.42，P=0.000），with increase of mineral calcium deposition in vitro. Furthermore，Wnt3a increased the mRNA level of Runx2（F=3 635.980，P=0.000；t（BMP9+Wnt3a）-BMP9=79.37，P=0.000；t（BMP9+Wnt3a）-Wnt3a=86.96，P=0.000）. Conclusion：Wnt3a，possibly through Runx2，acts synergistically on BMP9 induced osteogenic differentiation of MSCs.
