ObjectiveTo explore the effects on osteogenic differentiation of adipose derived stem cells (ADSCs) by simultaneously down-regulating Noggin combined with up-regulating bone morphogenetic protein 14 (BMP-14) in vitro. MethodsPrimary ADSCs were isolated and expanded in vitro from 5 Sprague Dawley rats (weighing, 250-300 g). ADSCs were transfected with lentiviral (Lv)-enhanced green fluorescent protein in group A (control group), with Lv-BMP-14 in group B, and with Lv-BMP-14 and Lv-Noggin shRNA in group C. BMP-14 and osteogenesis-related genes[collagen type I, alkaline phosphatase (ALP), and osteocalcin (OCN)] mRNA expression levels were detected by real time fluorescence quantitative PCR at 3, 7, and 14 days after transfection. Alizarin red staining for calcium nodules was also employed to assess the osteogenic ability of co-transfected ADSCs. ResultsAt 3 days after transfection, no significant difference was found in BMP-14 mRNA expression among groups P>0.05). At 7 and 14 days after transfection, BMP-14 mRNA expression was significantly higher in group C than groups A and B, and in group B than group A (P<0.05). At 3 days after transfection, collagen type I, ALP, and OCN mRNA expressions of group C were significantly higher than those of groups A and B (P<0.05), but no significant difference was shown between groups A and B P>0.05). At 7 and 14 days, collagen type I, ALP, and OCN mRNA expressions were higher in group C than groups A and B, and in group B than group A, showing significant difference (P<0.05) except collagen type I mRNA expression at 7 days between groups A and B P>0.05). The results of alizarin red staining showed that the amount of calcium nodules presented an increased tendency in the order of group A, group B, and group C. ConclusionBMP-14 is capable of enhancing osteogenic differentiation of ADSCs. A combination of inhibiting Noggin gene expression and enhancing BMP-14 gene expression in ADSCs can significantly strengthen osteogenic differentiation capability, showing significant synergistic effect.
【摘要】 目的 探討骨形成蛋白-2（bone morphogenetic protein-2，BMP-2）對室管膜前下區（anterior subventricular zone，SVZa）神經干細胞DLX5表達的影響。 方法 體外培養SVZa神經干細胞，用BMP-2及其拮抗劑Noggin誘導SVZa神經干細胞，分別用免疫熒光染色和逆轉錄-聚合酶鏈反應（RT-PCR）檢測DLX5表達變化。 結果 BMP-2組SVZa神經干細胞DLX5蛋白表達和DLX5mRNA表達水平明顯高于對照組（Plt;0.05），且該效應能被其拮抗劑Noggin特異性地抑制。 結論 BMP-2是DLX5上游調節基因，可促進SVZa神經干細胞DLX5的表達。【Abstract】 Objective To investigate the effect of bone morphogenetic protein-2 (BMP-2)on expression of DLX5 of neural stem cells in anterior subventricular zone (SVZa). Methods The neural stem cells of SVZa were separated and cultured in vitro, which were induced by BMP-2 and Noggin.Immunofluorescence staining and RT-PCR were employed to assay the expression of DLX5. Results The percentages of expression of DLX5 protein and DLX5 mRNA in BMP-2 group were much higher than those in the control group (Plt;0.05). And this induction could be specifically blocked by Noggin. Conclusion BMP-2 is an upstream gene of DLX5; BMP-2 can promote the expression of DLX5 of the neural stem cells of SVZa.