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目的探讨经典Wnt信号通路关键节点β-catenin对骨形态发生蛋白9(bone morphogenetic protein 9,BMP9)诱导间充质干细胞(mesenchymal stem cells,MSCs)成骨分化的影响。方法用重组腺病毒介导BMP9在C3H10T1/2细胞中过表达,联用β-catenin重组腺病毒上调β-catenin的表达,并通过RNA干扰抑制β-catenin的表达。分析C3H10T1/2细胞碱性磷酸酶(alkaline phosphatase,ALP)活性的变化;RT-PCR检测细胞成骨分化相关基因骨桥蛋白(osteopontin,OPN)和骨钙蛋白(osteocalcin,OC)基因mRNA的转录水平;茜素红S染色检测细胞的钙盐沉积。结果 BMP9单独作用能诱导C3H10T1/2细胞向成骨方向分化,并增强细胞ALP活性;单独的β-catenin无成骨诱导作用,但可剂量依赖性地增强BMP9诱导的C3H10T1/2细胞的ALP活性,并促进BMP9诱导的细胞OPN和OC基因mRNA的转录水平及钙盐沉积;抑制β-catenin表达可显著降低BMP9诱导的C3H1OT1/2细胞的ALP活性(P<0.05),下调OPN和OC基因mRNA的转录水平,并抑制钙盐沉积。结论经典Wnt信号通路可能通过β-catenin协同BMP9诱导C3H10T1/2细胞成骨分化,且BMP9诱导的成骨分化可能需要通过Wnt/β-catenin途径来实现。
Objective To investigate the effects of β-catenin, a key Wnt signaling pathway, on osteogenic differentiation of bone marrow mesenchymal stem cells (MSCs) induced by bone morphogenetic protein 9 (BMP9). Methods The recombinant adenovirus was used to overexpress BMP9 in C3H10T1 / 2 cells. The recombinant adenovirus was used to up-regulate the expression of β-catenin and inhibit the expression of β-catenin through RNA interference. The alkaline phosphatase (ALP) activity of C3H10T1 / 2 cells was analyzed. RT-PCR was used to detect the mRNA transcription of osteopontin (OPN) and osteocalcin (OC) Alizarin red S staining cells calcium deposition. Results BMP9 alone induced C3H10T1 / 2 cells to differentiate into osteoblasts and enhanced ALP activity. Β-catenin alone did not induce osteogenesis but increased the ALP activity of C3H10T1 / 2 cells induced by BMP9 in a dose-dependent manner , And promoted BMP9-induced OPN and OC gene mRNA transcription and deposition of calcium. Inhibition of β-catenin expression could significantly decrease the ALP activity of C3H1OT1 / 2 cells induced by BMP9 (P <0.05) Transcriptional level and inhibit the deposition of calcium salts. Conclusion The classical Wnt signaling pathway may induce osteogenic differentiation of C3H10T1 / 2 cells through β-catenin in combination with BMP9, and BMP9-induced osteogenic differentiation may require Wnt / β-catenin pathway.