成骨细胞条件性视黄酸信号失活小鼠模型的构建与验证  

Generation and validation of the conditional osteoblast-specific retinoic acid signaling inhibition mouse model

作　　者：孙思远 刘媛琪 崔怡雯 黄紫晗 梅李 代庆刚[4] 江凌勇[1,2] SUN Siyuan;LIU Yuanqi;CUI Yiwen;HUANG Zihan;MEI Li;DAI Qinggang;JIANG Lingyong(Department of Oral and Maxillofacial Surgery,Shanghai Ninth People's Hospital,Shanghai Jiao Tong University School of Medicine,College of Stomatology,Shanghai Jiao Tong University,National Center for Stomatology,National Clinical Research Center for Oral Diseases,Shanghai Key Laboratory of Stomatology,Shanghai Research Institute of Stomatology,Shanghai 200011,China;Shanghai-New Zealand Joint Laboratory of Dentistry,Centre for Clinical and Fundamental Craniofacial Research,Shanghai Ninth People's Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200011,China;Department of Oral Sciences,Faculty of Dentistry,University of Otago,Dunedin 9016,New Zealand;Department of 2nd Dental Centre,Shanghai Ninth People's Hospital,Shanghai Jiao Tong University School of Medicine,College of Stomatology,Shanghai Jiao Tong University,National Center for Stomatology,National Clinical Research Center for Oral Diseases,Shanghai Key Laboratory of Stomatology,Shanghai Research Institute of Stomatology,Shanghai 200011,China)

机构地区：[1]上海交通大学医学院附属第九人民医院口腔颅颌面科,上海交通大学口腔医学院,国家口腔医学中心,国家口腔疾病临床医学研究中心,上海市口腔医学重点实验室,上海市口腔医学研究所,上海200011 [2]上海-新西兰口腔医学联合实验室/口腔颅颌面基础与临床研究中心,上海交通大学医学院附属第九人民医院,上海200011 [3]新西兰奥塔哥大学牙学院正畸科,达尼丁9016 [4]上海交通大学医学院附属第九人民医院口腔第二门诊部,上海交通大学口腔医学院,国家口腔医学中心,国家口腔疾病临床医学研究中心,上海市口腔医学重点实验室,上海市口腔医学研究所,上海200011

出　　处：《上海交通大学学报（医学版）》2024年第6期676-686,共11页Journal of Shanghai Jiao tong University:Medical Science

基　　金：上海交通大学医学院“双百人”项目(20221809);中央高校基本科研业务费专项资金(YG2023ZD14);国家自然科学基金(82071083,82271006);上海市自然科学基金(21ZR1436900,22ZR1436700);海南省自然科学基金(824MS152);上海市科技创新行动计划国际科技合作项目/政府间国际科技合作项目(23410713600);上海交通大学医学院附属第九人民医院交叉研究基金(JYJC202116);上海交通大学医学院生物材料与再生医学交叉研究项目(2022LHB02);上海交通大学医学院附属第九人民医院原创项目(JYYC003)。

摘　　要：目的·构建并验证可模拟维生素A缺乏症(vitamin A deficiency,VAD)导致的颅颌面骨畸形且出生后可存活的模型小鼠。方法·运用Cre-LoxP重组酶系统,通过将Osx^(Cre)与Rosa26dnRARα/dnRARα基因型的小鼠多代杂交,构建成骨细胞视黄酸受体α显性负突变体(dominant-negative retinoid acid receptorαmutation,dnRARα)条件性过表达小鼠Osx^(Cre);Rosa26^(dn/dn),实现成骨细胞视黄酸信号条件性失活以模拟VAD状态。取Osx^(Cre);Rosa26^(dn/dn)小鼠及其同窝对照小鼠股骨骨髓间充质干细胞(bone mesenchymal stem cell,BMSC)与颅顶骨细胞,成骨诱导后,采用蛋白质印迹法(Western blotting)验证成骨细胞中视黄酸受体α(retinoid acid receptorα,RARα)蛋白的表达水平。取Osx^(Cre);Rosa26^(dn/dn)小鼠及其同窝对照小鼠颅顶骨细胞经诱导形成的成骨细胞,采用双荧光素酶报告基因技术验证视黄酸信号通路抑制程度。同时分别应用表达Cre、eGFP的腺病毒(Ad-eGFP与Ad-Cre)感染Rosa26^(dn/dn)小鼠股骨BMSC与颅顶骨细胞,成骨诱导后运用相同方法评价显性负突变蛋白表达水平与视黄酸信号通路抑制程度。取6周龄小鼠颅骨,运用Micro-CT及三维重建技术验证Osx^(Cre);Rosa26^(dn/dn)模型小鼠颅颌面骨畸形表型。结果·Western blotting结果显示,Osx^(Cre);Rosa26^(dn/dn)小鼠股骨及颅顶骨成骨细胞相较同窝对照小鼠表现出RARα蛋白水平上调。双荧光素酶报告基因实验结果显示Osx^(Cre);Rosa26^(dn/dn)小鼠成骨细胞的视黄酸反应元件(retinoid acid response element,RARE)活性相较同窝对照小鼠下调(P<0.05)。同时,Ad-Cre感染后Rosa26^(dn/dn)小鼠股骨及颅顶骨成骨细胞相较Ad-eGFP感染后表现出RARα蛋白水平上调,成骨细胞的RARE活性下调(P<0.05)。Micro-CT及三维重建结果显示,6周龄Osx^(Cre);Rosa26^(dn/dn)小鼠颅骨呈现长度缩短、骨发育不全等VAD样颅颌面骨畸形。结论·成功构建了成骨细胞条件性视黄酸信号失活�Objective·To construct and verify the mouse model that can mimic the vitamin A deficiency(VAD)-like craniofacial skeletal deformity and do not cause embryonic death.Methods·Based on the Cre-LoxP system,the Osx^(Cre);Rosa26^(dn/dn)mice expressing osteoblast-specific dominant-negative retinoid acid receptorα(dnRARα)mutation were obtained by hybridization through Osx^(Cre)and Rosa26dnRARα/dnRARαmice,to achieve the conditional inhibition of retinoic acid signaling to simulate VAD disease.Femur bone mesenchymal stem cells(BMSCs)and parietal bone cells of Osx^(Cre);Rosa26^(dn/dn)mice and their control littermates were isolated and underwent osteogenic induction,to assess the expression of retinoid acid receptorα(RARα)protein through Western blotting.Osteoblasts induced from parietal bone cells of Osx^(Cre);Rosa26^(dn/dn)mice and their control littermates were isolated and the effect of retinoic acid signaling inhibition was verified through dual luciferase gene reporter assay.Meanwhile,Ad-eGFP or Ad-Cre adenovirus-infected femur BMSCs and parietal bone cells of Rosa26^(dn/dn)mice underwent osteogenic induction to assess the expression of dominant-negative mutant protein and the inhibition of the retinoic acid signaling pathway in vitro by Western blotting and dual luciferase gene reporter assay.Moreover,the skulls of 6-week-old Osx^(Cre);Rosa26^(dn/dn)mice were collected,and Micro-CT scanning and three-dimensional(3D)reconstruction were performed to verify the craniofacial skeletal deformities of the mouse model.Results·Western blotting results demonstrated that the level of RARαprotein increased in the femur and parietal osteoblasts of Osx^(Cre);Rosa26^(dn/dn)mice compared to that of their control littermates,and also increased in the Ad-Cre-infected femur and parietal osteoblasts of Rosa26^(dn/dn)mice compared to that in the Ad-eGFP-infected group(P<0.05).Dual luciferase gene reporter assay results indicated that the activity of retinoid acid response element(RARE)was inhibited in the osteoblasts of Osx^(Cr

关 键 词：维生素A缺乏症 颅颌面骨畸形 成骨细胞 显性负突变基因条件性表达小鼠 视黄酸受体 

分 类 号：R78[医药卫生—口腔医学]