Shh信号参与调控BMP9诱导的间充质干细胞成骨分化

Shh Signaling is Involved in Regulating BMP9-induced Osteogenic Differentiation of Mesenchymal Stem Cells

目的:观察sonic hedgehog(Shh)信号通路在骨形态发生蛋白9(BMP9)诱导的小鼠间充质干细胞(MSCs)C3H10T1/2和C2C12成骨分化中的作用,并初步探讨其作用机制。方法:Shh信号通路抑制剂Cyclopamine和激活剂Purmorphamine以及过表达Shh腺病毒分别作用于BMP9处理的C3H10T1/2和C2C12细胞,碱性磷酸酶(ALP)检测早期成骨指标ALP,茜素红S染色检测晚期成骨指标钙盐沉积,RT-PCR检测Shh信号相关基因以及成骨关键转录因子的表达,Western blot检测Shh的表达,荧光素酶报告基因检测Smad1/5/8的转录调控活性。结果:BMP9促进Shh信号相关基因的表达,激活Shh信号可增强BMP9诱导的C3H10T1/2和C2C12细胞早晚期成骨分化并促进了BMP9诱导的Smad荧光素酶活性,抑制Shh信号后作用相反。结论:激活Shh信号通路可促进BMP9诱导的小鼠MSCs成骨分化,抑制其活性后作用相反。

Objective： To analysis the effect of Sonic hedgehog signaling on BMP9-induced osteogenic differentiation of mesenchymal stem cells and the mechanism involved. Methods： C3H10T1/2 and C2C12 cells were treated with Cyclopamine, Purmorphamine, adenovirus Shh or/and BMP9, then the early osteogenic marker ALP activity was detected by quantitative and staining assay, the later osteogenic marker calcium deposition was detected by Alizarin Red S were detected by RT-PCR, staining, the expressions of Shh signaling related genes and osteogenesis related genes the expressions of Shh detected by Western blot, luciferae reporter assay was detected by quantitative. Results： BMP9 can promote the expressions of Shh signaling related genes. Purmorphamine and adenovirus Shh can increase the ealy and later osteogenic differentiation and expressions of osteogenesis key tromscription factors of C3H10T1/2 and C2C12 cells induced by BMP9, but Cyclopamine has the opposite effects. Furthermore, Purmorphamine and adenovirus Shh also led to luciferase activity of canonical Smad pathway stimulated by BMP9. Conclusion： Shh signaling influence BMPg-induced osteogenic differentiation of C3H10T1/2 and C2C12 mesenchymal stem cells.