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以骨形态发生蛋白9(bone morphogenetic protein 9,BMP9)作为诱导小鼠间充质干细胞C3H10T1/2定向成骨分化的细胞因子,观察过表达S100A6对成骨分化的影响。用重组腺病毒AdBMP9与AdS100A6共感染C3H10T1/2细胞,随后检测成骨分化标志物,包括Runx2、碱性磷酸酶(alkaline phosphatase,ALP)、骨桥素(osteopontin,OPN)和钙盐沉积;检测方法包括免疫细胞化学、ALP染色、活性分析以及茜素红S染色;同时,用Western blot检测β-catenin的表达。过表达S100A6对所诱导的间充质干细胞C3H10T1/2成骨分化的早期指标Runx2的蛋白水平无明显影响(P>0.05;ICC法),对第7天的ALP和第18天钙盐的沉积也无明显影响(P>0.05);但是,能够引起第12天的OPN升高(P<0.05);同时,S100A6对BMP9诱导的C3H10T1/2细胞中的β-catenin水平的影响在第3、7天不明显(P>0.05),但在第12天时可致β-catenin水平下调(P<0.05)。过表达S100A6可促进BMP9诱导的C3H10T1/2细胞中OPN的表达,但对最终的成骨分化无明显影响。
Bone morphogenetic protein 9 (BMP9) was used as a cytokine to induce osteogenic differentiation of mouse mesenchymal stem cells C3H10T1 / 2, and the effect of overexpression of S100A6 on osteogenic differentiation was observed. C3H10T1 / 2 cells were co-infected with recombinant adenovirus AdBMP9 and AdS100A6, followed by detection of osteogenic differentiation markers including Runx2, alkaline phosphatase (ALP), osteopontin (OPN) Methods Immunocytochemistry, ALP staining, activity analysis and alizarin red S staining; the same time, Western blot detection of β-catenin expression. Overexpression of S100A6 had no significant effect on the protein level of Runx2, an early indicator of osteogenic differentiation of mesenchymal stem cells (C3H10T1 / 2) induced by ICC (P> 0.05); on day 7 ALP and on day 18 calcium deposition (P <0.05). However, it increased the OPN on the 12th day (P <0.05). At the same time, the effect of S100A6 on the β-catenin level in BMP9-induced C3H10T1 / 2 cells was not obvious at the 3rd, 7 days was not obvious (P> 0.05), but on the 12th day, the level of β-catenin was down-regulated (P <0.05). Overexpression of S100A6 promoted the expression of OPN in C3H10T1 / 2 cells induced by BMP9, but had no significant effect on the final osteogenic differentiation.