DLL1在骨形态形成蛋白9诱导骨髓间充质干细胞成骨分化中的作用  

Delta-like 1 promotes osteogenic differentiation of mesenchymal stem cells induced by BMP9

作　　者：廉静[1] 杨伦韵 曹俊杰[1] 罗进勇[1] 何百成[2] 唐敏[1] 

机构地区：[1]重庆医科大学检验医学院临床检验诊断学教育部重点实验室,重庆400016 [2]重庆医科大学生物化学与药理学重点实验室,重庆2400016

出　　处：《第三军医大学学报》2016年第1期38-43,共6页Journal of Third Military Medical University

基　　金：重庆市自然科学基金(CSTC2012jj A10004);重庆市教委科学技术研究项目(KJ130305)~~

摘　　要：目的探讨Notch配体Delta-like1(DLL1)在骨形态形成蛋白9(bone morphogenetic proteins 9,BMP9)诱导的骨髓间充质干细胞(mesenchymal stem cells,MSCs)成骨分化中的作用及机制。方法利用DLL1重组腺病毒上调永生化小鼠胚胎成纤维细胞(immortalized mouse embryonic fibroblasts,i MEF)中DLL1 mRNA的表达,分别采用细胞化学染色、活性测定对早期成骨指标碱性磷酸酶(alkaline phosphatase,ALP)和晚期成骨指标钙盐沉积的影响;其次裸鼠皮下异位成骨实验进一步了解DLL1的体内成骨作用;最后用qRT-PCR、Western blot和荧光素酶检测DLL1对BMP9经典信号通路中Ⅰ型受体、Smad1/5/8磷酸化和Smad结合元件(smad-binding element,SBE)转录活性影响。结果与对照组相比,DLL1在体外能明显促进BMP9诱导的MSCs早期成骨指标ALP活性和晚期成骨指标钙盐沉积的生成;同时也能明显促进BMP9诱导的MSCs裸鼠皮下异位成骨作用;BMP9信号通路中ALK2表达明显增加,而对ALK1则没有影响;Smad1/5/8蛋白量没有明显变化,而p-Smad1/5/8蛋白及SBE活性明显增加(P<0.05)。结论 DLL1可以促进BMP9诱导的MSCs成骨分化,可能通过影响BMP9信号通路来实现其促成骨作用。Objectives To determine the role of Notch ligand,Delta-like 1（ DLL1） in bone morphogenetic proteins 9（ BMP9）-mediated osteogenesis in mesenchymal stem cells（ MSCs） and investigate its possible mechanisms. Methods Recombinant adenoviruses were used to over-express DLL1 in immortalized mouse embryonic fibroblasts（ i MEF）. Cytochemical staining was used to evaluate the early-stage osteogenetic index,alkaline phosphatase（ ALP） and the late-stage osteoblastic indicator,calcium deposits.Ectopic bone formation assay was conducted in nude mice,then HE staining and micro-CT scanning were employed to analyze osteogenetic status so as to determine the role of DLL1 in the process. qRT-PCR,Western blotting and luciferase reporter assay were conducted to detect the expression levels of BMP receptors,pSmad1 /5 /8,and activity of Smad-binding element（ SBE）. Results Compared with control group,DLL1 in vitro treatment obviously enhanced the osteogenic indicators ALP and calcium deposits,and in vivo promoted ectopic bone formation in nude mice. ALK2 in BMP signal pathway was significantly elevated,but no change was seen in the level of ALK1. There were also no obvious changes in the total protein levels of Smad1 /5 /8,but those of phosphorylated Smad1 /5 /8 were increased. SBE activity was also enhanced（ P 0. 05）.Conclusion DLL1 enhances BMP-induced osteogenesis in MSCs both in vitro and in vivo,and it might exert the effect through BMP-Smad pathway.

关 键 词：间充质干细胞 Delta-like1 骨形态发生蛋白9 NOTCH信号通路 

分 类 号：R322.71[医药卫生—人体解剖和组织胚胎学] R329.21[医药卫生—基础医学]