Smad信号通路在骨形成蛋白9促进牙囊干细胞成骨向分化中的研究  

Smad signaling pathway regulates bone morphogenetic protein 9-induced osteogenic differentiation of rat dental follicle stem cells in vitro

作　　者：任格 聂利 杨霞[1] 黄丽娜[1] 王明[1] 邱叶[1] 李洁仪[1] 李丛华[1] REN Ge;NIE Li;YANG Xia;HUANG Lina;WANG Ming;QIU Ye;LI Jieyi;LI Conghua(Chongqing Key Laboratory of Oral Disease and Biomedical Sciences, Chongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education, Stomatological Hospital of Chongqing Medical University, Chongqing, 401147,China)

机构地区：[1]重庆医科大学附属口腔医院口腔疾病与生物医学重庆市重点实验室重庆市高校市级口腔生物医学工程重点实验室,重庆401147

出　　处：《第三军医大学学报》2018年第12期1066-1072,共7页Journal of Third Military Medical University

基　　金：重庆市卫计委重点项目(2012-1-056);重庆市卫计委面上项目(2016MSXM049);重庆市科委自然科学基金(CSTC 2016jcyj A0415)~~

摘　　要：目的探索骨形成蛋白9(bone morphogenetic protein 9,BMP9)在诱导大鼠牙囊干细胞(rat dental follicle stem cells,r DFCs)成骨向分化过程中的Smad信号通路调控机制。方法重组腺病毒骨形成蛋白9(recombinant adenoviruses expressing BMP9,Ad-BMP9)转染纯化的第3代r DFCs后,Realtime q PCR、碱性磷酸酶(alkaline phosphatase,ALP)染色及活性检测观察BMP9调节r DFCs早期及中晚期成骨能力,茜素红S染色检测钙盐沉积,Western blot检测Smad1/5/8磷酸化水平。结果 BMP9促进r DFCs成骨因子表达:成骨转录因子Runx2、成骨细胞特异性转录因子Osterix、ALP及骨桥蛋白(osteopontin,OPN)在BMP9刺激组较空白组均明显升高(P<0.05),钙盐沉积及钙结节形成也明显多于空白组,且BMP9促进了Smad1/5/8蛋白磷酸化水平升高。结论 BMP9通过激活Smad信号通路,提高Smad1/5/8蛋白磷酸化水平从而促进早中晚期成骨因子及碱性磷酸酶表达,促进了r DFCs成骨向分化。ObjectiveTo investigate the role of Smad signaling pathway in the regulatory mechanism of bone morphogenetic protein 9 （BMP9）induced osteogenic differentiation of rat dental follicle stem cells （rDFCs）. MethodsThe third passage of purified rDFCs were transfected with the recombinant adenoviruses expressing BMP9 （AdBMP9）, and the early and late osteogenesis of rDFCs was detected using real-time qPCR, alkaline phosphatase （ALP） staining, ALP activity assay and Alizarin red S staining. The phosphorylation of Smad1/5/8 in the transfected cells was determined using Western blotting. ResultsCompared with the control cells, AdBMP9transfected rDFCs showed significantly enhanced expression of the osteogenic factors including Runx2, Osterix, ALP and osteopontin（OPN） （P〈0.05） with also increased calcium deposition and formation of calcium nodules in the early, intermediate and late stages of osteogenesis. Adenovirusmediated BMP9 gene transduction significantly increased the phosphorylation levels of Smad1/5/8 proteins in rDFCs. ConclusionBMP9 can promote the osteogenic differentiation of rDFCs by activating Smad signaling pathway and increasing the phosphorylation level of Smad1/5/8 proteins to enhance the expression of ALP and osteogenic factors in the early, intermediate and late stages of differentiation.

关 键 词：牙囊干细胞 骨形成蛋白9 成骨分化 SMAD信号通路 

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]