Liu Hao, Rui Yongjun, Sun Zhenzhong, Tang Fenglin, Ding Tao
Department of Orthopedics, Wuxi 9th People's Hospital, Wuxi Jiangsu, 214000, P. R. China.
Department of Orthopedics, Wuxi People's Hospital.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2016 Oct 8;30(10):1270-1275. doi: 10.7507/1002-1892.20160259.
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.
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.
At 3 days after transfection, no significant difference was found in BMP-14 mRNA expression among groups >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 (<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 (<0.05), but no significant difference was shown between groups A and B >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 (<0.05) except collagen type I mRNA expression at 7 days between groups A and B >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.
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.
在体外通过同时下调Noggin并上调骨形态发生蛋白14（BMP-14）来探究其对脂肪来源干细胞（ADSCs）成骨分化的影响。
从5只体重250 - 300 g的Sprague Dawley大鼠体外分离并扩增原代ADSCs。A组（对照组）用慢病毒（Lv）-增强型绿色荧光蛋白转染ADSCs，B组用Lv-BMP-14转染，C组用Lv-BMP-14和Lv-Noggin shRNA转染。在转染后3、7和14天，通过实时荧光定量PCR检测BMP-14和成骨相关基因[Ⅰ型胶原、碱性磷酸酶（ALP）和骨钙素（OCN）]的mRNA表达水平。还用茜素红染色检测钙结节来评估共转染ADSCs的成骨能力。
转染后3天，各组间BMP-14 mRNA表达无显著差异（>0.05）。转染后7天和14天，C组BMP-14 mRNA表达显著高于A组和B组，B组高于A组（<0.05）。转染后3天，C组Ⅰ型胶原、ALP和OCN mRNA表达显著高于A组和B组（<0.05），但A组和B组之间无显著差异（>0.05）。在7天和14天，C组Ⅰ型胶原、ALP和OCN mRNA表达高于A组和B组，B组高于A组，除A组和B组之间7天的Ⅰ型胶原mRNA表达外均有显著差异（<0.05）（>0.05）。茜素红染色结果显示，钙结节数量按A组、B组、C组的顺序呈增加趋势。
BMP-14能够增强ADSCs的成骨分化。在ADSCs中抑制Noggin基因表达并增强BMP-14基因表达的联合作用可显著增强成骨分化能力，显示出显著的协同效应。
