激活骨髓基质细胞Notch信号体外促进小鼠成骨作用

Activation of Notch signaling in bone marrow stromal cells promotes osteogenesis in mice in vitro

目的探究体外激活骨髓基质细胞(BMSC)缺刻基因(Notch)信号对成骨分化的影响。方法使用Cre重组腺病毒(Ad-Cre)以及对照腺病毒(Ad-GFP),分别感染Notch1-NICD^(flox/flox)小鼠BMSC,分为Ad-Cre组和Ad-GFP组。Western blot法检测Notch1-NICD蛋白表达水平;碱性磷酸酶(ALP)染色及生化定量检测早期成骨分化水平,茜素红S染色及矿化定量检测晚期钙盐沉积水平;实时荧光定量PCR检测Notch靶基因发状分裂增强子1(Hes1)、含YRPW基序的bHLH转录因子Hes相关家族1(Hey1)、Hey2、HeyL、成骨标志基因ALP、Runt相关转录因子2(RUNX2)、骨转化因子(Osx)、骨钙素(Ocn)以及促血管生成因子血管内皮生长因子(VEGF)、缺氧诱导因子1α(HIF-1α)的表达。结果Ad-Cre成功激活Notch1-NICD^(flox/flox)小鼠BMSC中的Notch信号;Ad-Cre组的ALP活性相较Ad-GFP组明显升高,晚期钙盐沉积水平明显升高;ALP、RUNX2、Osx、Ocn和VEGF、HIF-1α的表达显著增加。结论体外激活小鼠BMSC的Notch信号显著促进成骨分化。

Objective To investigate the osteogenic differentiation of bone marrow stromal cells(BMSC)with Notch signaling activation in vitro.Methods The BMSC derived from Notch1-NICD^(flox/flox)mice were infected with recombinant adenovirus expressing Cre or GFP respectively,and designated as Ad-Cre group and Ad-GFP group.The expression of Notch1 was evaluated by Western blot analysis.Alkaline phosphatase(ALP)activity was determined by ALP staining and biochemical quantification,and the calcium deposition was analyzed with Alizarin red S staining.Real-time fluorescence quantitative PCR(qPCR)was used to quantify the expression of Notch target genes(Hes1,Hey1,Hey2,HeyL),osteogenic differentiation-related genes(ALP,RUNX2,osterix,osteocalcin),and angiogenesis factors(VEGF,HIF-1α).ResultsNotch signaling in BMSC of Notch1-NICD^(flox/flox)mice was activated successfully by Ad-Cre,as was evidenced by the significantly elevated expression of Notch1 and Notch target genes.Compared with the Ad-GFP group,ALP activity and the late calcium deposition were significantly increased upon treatment with Ad-Cre.qPCR results demonstrated that the expression of ALP,RUNX2,osterix,osteocalcin,VEGF,HIF-1αin the Ad-Cre group were significantly upregulated.Conclusion Activation of Notch signaling in BMSC in vitro significantly promotes osteogenic differentiation.