Objective: To 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.
Methods: Primary 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.
Results: At 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.
Conclusions: BMP-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.
目的: 在体外环境下同时下调细胞中Noggin基因和增加BMP-14基因，观察对脂肪来源干细胞（adipose derived stem cells，ADSCs）成骨分化能力的影响。.
方法: 取健康成年SD大鼠5只，体质量250～300 g，采用Ⅰ型胶原酶消化法获取原代ADSCs，体外培养、扩增。使用慢病毒载体将目的基因转染大鼠ADSCs，根据目的基因不同将实验分为3组，A组为空载体病毒Lv-增强型绿色荧光蛋白转染对照组，B组为Lv-BMP-14转染组，C组为BMP-14+Noggin shRNA转染组。分别于转染后3、7、14 d，采用实时荧光定量PCR检测BMP-14及成骨相关基因[Ⅰ型胶原、ALP、骨钙素（osteocalcin，OCN）]的表达，转染后14 d采用茜素红染色鉴定其成骨分化能力。.
结果: 转染后3 d，各组BMP-14 mRNA相对表达量比较差异无统计学意义（P＞0.05）；7、14 d，C组BMP-14 mRNA相对表达量高于A、B组，B组高于A组，比较差异均有统计学意义（P＜0.05）。转染后3 d，C组各成骨相关基因mRNA相对表达量显著高于A、B组（P＜0.05）；A、B组间比较差异无统计学意义（P＞0.05）。转染后7、14 d，C组各成骨相关基因mRNA相对表达量显著高于A、B组，B组高于A组，除转染后7 d A、B组间Ⅰ型胶原mRNA相对表达量比较差异无统计学意义（P＞0.05）外，其余各组间比较差异均有统计学意义（P＜0.05）。转染后14 d茜素红染色示，A、B、C组钙结节量呈递增趋势。.
结论: BMP-14具有增强大鼠ADSCs向成骨细胞分化的能力；沉默细胞中的Noggin基因、联合BMP-14基因共同作用于大鼠ADSCs，其体外成骨分化能力明显强于BMP-14单基因转染，两者有显著的协同作用。.
Keywords: Adipose derived stem cells; Bone morphogenetic protein 14; Co-transfection; Lentivirus; Noggin; Rat.