【作者】 张雨垚； 钟旖； 于金华；

【Author】 Yuyao Zhang;Yi Zhong;Jinhua Yu;Department of Endodontics, The Affiliated Stomatological Hospital of Nanjing Medical University;Jiangsu Province Key Laboratory of Oral Diseases, Nanjing Medical University;Jiangsu Province Engineering Research Center of Stomatological Translational Medicine;Department of Oral Pathology, The Affiliated Stomatological Hospital of Nanjing Medical University;

【机构】 南京医科大学附属口腔医院牙体牙髓病科； 江苏省口腔疾病研究重点实验室； 江苏省口腔转化医学工程研究中心； 南京医科大学附属口腔医院口腔病理科；

【摘要】 目的:本研究旨在探讨序列相似性为83的家族成员A (family with sequence similarity 83 member A,FAM83A)对人牙髓干细胞成牙和成骨分化的作用及可能的分子机制。方法:酶消化法分离培养人牙髓干细胞(human dental pulp stem cells, hDPSCs)。通过流式细胞术、成脂、成软骨及成骨诱导体外鉴定hDPSCs表型特点及生物学特性。利用小干扰RNA沉默hDPSCs的FAM83A表达后,通过qRT-PCR和Western blot检测人牙髓干细胞成牙/成骨向分化相关基因牙本质涎磷蛋白(dentin sialophosphoprotein,DSPP)、牙本质基质蛋白1(dentine matrix protein 1,DMP1)、Runt相关转录因子2 (Runt-related transcription factor 2,RUNX2)以及成骨细胞特异性转录因子Osterix(Osx)的表达。通过Western blot检测Wnt/β-catenin信号通路蛋白β-catenin和p-β-catenin蛋白的表达变化。通过碱性磷酸酶染色和茜素红S染色观察FAM83A对hDPSCs矿化能力的影响。结果:分离培养的hDPSCs来源于间充质组织并且具有克隆形成和多向分化能力。沉默FAM83A后,qRT-PCR和Western blot结果表明人牙髓干细胞成牙/成骨向分化相关基因DSPP、 DMP1、RUNX2及Osx在mRNA和蛋白水平的的表达均显著下调; Western blot结果表明,沉默FAM83A后hDPSCs的Wnt/β-catenin信号通路蛋白β-catenin和p-β-catenin的表达也显著下调;碱性磷酸酶染色和茜素红S染色结果表明,沉默FAM83A后hDPSCs的矿化能力受到抑制。结论:本研究表明FAM83A可能通过Wnt/β-Catenin信号通路调控人牙髓干细胞再生牙髓-牙本质复合体的能力,为临床牙髓损伤修复机制研究提供了新的见解。更多还原

【Abstract】 Objective: To detect the effects of family with sequence similarity 83 member A(FAM83A) on the odontogenic and osteogenic differentiation of human dental pulp stem cells and the potential mechanism.Methods : The human dental pulp stem cells(hDPSCs) were isolated by enzymatic digestion. The phenotypic and biological characteristics of hDPSCs were identified by flow cytometry, lipogenesis, chondrogenesis and osteogenesis induction in vitro. After silencing the expression of FAM83A of hDPSCs by small interfering RNA, qRT-PCR and Western blot were used to detect the expressions of dentin sialophosphoprotein(DSPP), dentine matrix protein 1(DMP1), Runt-related transcription factor 2(RUNX2) and osteoblast-specific transcription factor, Osterix(Osx) of hDPSCs. The expressions of β-catenin and p-β-catenin were also detected by Western blot. The effects of FAM83A on the mineralization ability of hDPSCs were determined by alkaline phosphatase staining and alizarin red S staining.Results:The isolated hDPSCs were derived from mesenchymal tissues and possessed the ability of clone formation and multidirectional differentiation. The results of qRT-PCR and Western blot showed that the expressions of DSPP, DMP1, RUNX2 and Osx were significantly downregulated at both mRNA and protein levels of hDPSCs after silencing FAM83A. The results of Western blot showed that the expression ofβ-catenin and p-β-catenin in hDPSCs were also significantly downregulated after silencing FAM83A. The results of alkaline phosphatase staining and alizarin red S staining showed that the mineralization ability of hDPSCs was inhibited after silencing FAM83A.Conclusion : FAM83A can regulate the the odontogenic and osteogenic differentiation through Wnt/β-catenin signaling pathway.更多还原