体外激活骨细胞Notch信号对骨髓基质细胞成骨分化的影响  

Impact of activating Notch signaling in osteocytes on osteogenic differentiation of bone marrow stromal cells in vitro

作　　者：谢映春 涂小林[1] XIE Yingchun;TU Xiaolin(Bone Development and Regeneration Platform,Life Sciences Institute,Chongqing Medical University,Chongqing,400016,China)

机构地区：[1]重庆医科大学生命科学研究院骨发育与再生平台,重庆400016

出　　处：《第三军医大学学报》2020年第9期891-898,共8页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(81672118)。

摘　　要：目的探究体外激活骨细胞Notch信号对骨髓基质细胞(bone marrow stromal cells,BMSCs)成骨分化的影响。方法设置Cre重组腺病毒(Ad-Cre)及对照腺病毒GFP(Ad-GFP),分别转染RosaNotch小鼠骨细胞后与野生C57BL/6小鼠BMSCs共培养为Ad-Cre组及Ad-GFP组,设置仅BMSCs为Blank组。碱性磷酸酶染色及生化定量测定碱性磷酸酶(ALP);qPCR检测转染后骨细胞Notch信号靶基因及共培养产物成骨相关标志物、Notch信号配体及成血管相关标志物,茜素红染色检测成骨诱导21 d钙盐沉积水平。结果Ad-Cre成功激活RosaNotch小鼠骨细胞Notch信号,ALP染色及生化定量结果显示Ad-Cre组ALP表达相较Ad-GFP组及Blank组显著降低(P<0.05),Ad-GFP组与Blank组间无统计学差异;qPCR结果显示Ad-Cre组共培养产物成骨标志物ALP、核心结合因子a1(Runx2)、特异性骨转化因子(Ostrix)及Notch配体Dll4 mRNA转录水平相较Ad-GFP组及Blank组显著降低(P<0.05),Jag1 mRNA转录水平显著降低(P<0.05);血管内皮生长因子(VEGF)、低氧诱导因子(hypoxia-inducible factor 1α,HIF1α)、血小板内皮细胞黏附分子(platelet endothelial cell adhesion molecule-1,CD31/PECAM-1)及内皮粘蛋白(endomucin,EMCN)mRNA转录水平相较Ad-GFP组及Blank组显著增高(P<0.05);茜素红染色结果显示Ad-Cre组在成骨诱导培养基作用21 d后钙盐沉积水平相较Ad-GFP组及Blank组显著降低。结论体外激活骨细胞Notch信号抑制骨髓基质细胞成骨分化。Objective To investigate the effect and underlying mechanism of activating Notch signaling in osteocytes on osteogenic differentiation of bone marrow stromal cells(BMSCs).Methods The osteocytes derived from RosaNotch mice were infected with recombinant adenovirus expressing Cre or GFP respectively,and then the obtained cells were co-cultured with BMSCs isolated from wild-type C57 BL/6 mice,and named as Ad-Cre group and Ad-GFP group,with a BMSCs group without any treatment as blank control.Alkaline phosphatase(ALP)staining and detection of ALP relative activity were used to measure the expression of ALP.The mRNA expression levels of the target genes in osteocyte of RosaNotch and ligands of Notch signaling,osteogenic markers,and angiogenic makers in the co-cultured product were detected by qPCR.Alizarin red staining was applied to test the matrix mineralization on day 21.Results Infection of recombinant adenovirus Ad-Cre could successfully activate Notch signaling.ALP staining and detection of ALP activity showed that the expression level of ALP was significantly lower in the Ad-Cre group than the Ad-GFP group and Blank group(P<0.05),and there was no statistical difference in the level between the latter 2 groups.The results of qPCR indicated that the mRNA levels of osteogenic makers ALP,Osterix,Runx2 and Notch ligand Dll4 were deceased significantly,while those of Jag1,VEGF,hypoxia-inducible factor 1α(HIF1α),platelet endothelial cell adhesion molecule-1(CD31/PECAM-1),and endomucin(EMCN)were statistically increased in the Ad-Cre group when compared with the Ad-GFP group and Blank group(P<0.05).Alizarin red staining displayed that the Ad-Cre group had more calcium deposition in 21 d after co-culture than the Ad-GFP group and blank group(P<0.05).Conclusion Activating Notch signaling in osteocytes inhibits osteogenic differentiation of BMSCs in vitro.

关 键 词：骨细胞 NOTCH信号 骨髓基质细胞 成骨分化 

分 类 号：R322.71[医药卫生—人体解剖和组织胚胎学] R329.2[医药卫生—基础医学]