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目的探讨Notch配体Delta-like1(DLL1)在骨形态形成蛋白9(bone morphogenetic proteins 9,BMP9)诱导的骨髓间充质干细胞(mesenchymal stem cells,MSCs)成骨分化中的作用及机制。方法利用DLL1重组腺病毒上调永生化小鼠胚胎成纤维细胞(immortalized mouse embryonic fibroblasts,i MEF)中DLL1 mRNA的表达,分别采用细胞化学染色、活性测定对早期成骨指标碱性磷酸酶(alkaline phosphatase,ALP)和晚期成骨指标钙盐沉积的影响;其次裸鼠皮下异位成骨实验进一步了解DLL1的体内成骨作用;最后用qRT-PCR、Western blot和荧光素酶检测DLL1对BMP9经典信号通路中Ⅰ型受体、Smad1/5/8磷酸化和Smad结合元件(smad-binding element,SBE)转录活性影响。结果与对照组相比,DLL1在体外能明显促进BMP9诱导的MSCs早期成骨指标ALP活性和晚期成骨指标钙盐沉积的生成;同时也能明显促进BMP9诱导的MSCs裸鼠皮下异位成骨作用;BMP9信号通路中ALK2表达明显增加,而对ALK1则没有影响;Smad1/5/8蛋白量没有明显变化,而p-Smad1/5/8蛋白及SBE活性明显增加(P<0.05)。结论 DLL1可以促进BMP9诱导的MSCs成骨分化,可能通过影响BMP9信号通路来实现其促成骨作用。
Objective To investigate the role of Notch ligand Delta-like1 (DLL1) in osteogenic differentiation of bone marrow mesenchymal stem cells (MSCs) induced by bone morphogenetic proteins 9 (BMP9). Methods DLL1 recombinant adenovirus was used to up-regulate the expression of DLL1 mRNA in immortalized mouse embryonic fibroblasts (i MEF). Cytochemical staining and enzyme activity assay were used to detect the expression of alkaline phosphatase , ALP), and late-stage osteogenesis index calcium deposition. Secondly, the subcutaneous heterotopic osteogenesis experiment in nude mice further revealed the in vivo osteogenesis effect of DLL1. Finally, qRT-PCR, Western blot and luciferase detection of DLL1 on BMP9 classic signal Pathway, type I receptor, Smad1 / 5/8 phosphorylation and smad-binding element (SBE) transcriptional activity. Results Compared with the control group, DLL1 could significantly promote BMP9-induced ALP activity in early osteosynthesis and calcium deposition in advanced osteosynthesis-induced MSCs, and significantly promote the subcutaneous heterotopic osteogenesis of BMP9-induced MSCs in nude mice (P <0.05). The expression of ALK2 in BMP9 signal pathway was significantly increased, but not in ALK1. The protein level of Smad1 / 5/8 did not change significantly, but the activity of p-Smad1 / 5/8 protein and SBE increased significantly. Conclusion DLL1 can promote the osteogenic differentiation of MSCs induced by BMP9, which may promote bone formation by affecting the BMP9 signaling pathway.