成骨细胞条件性视黄酸信号失活小鼠模型的构建与验证

doi:10.3969/j.issn.1674-8115.2024.06.002

上海交通大学学报（医学版） ›› 2024, Vol. 44 ›› Issue (6): 676-686.doi: 10.3969/j.issn.1674-8115.2024.06.002

成骨细胞条件性视黄酸信号失活小鼠模型的构建与验证

孙思远¹^,²(), 刘媛琪¹^,²(), 崔怡雯¹^,², 黄紫晗¹^,², 梅李²^,³, 代庆刚⁴, 江凌勇¹^,²()

^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

收稿日期:2024-02-01 接受日期:2024-03-21 出版日期:2024-06-28 发布日期:2024-06-28
通讯作者: 江凌勇 E-mail:ssy1412832053@163.com;liuyuanqi@sjtu.edu.cn;jianglingyong@sjtu.edu.cn
作者简介:孙思远（1998—），男，硕士生；电子信箱：ssy1412832053@163.com
刘媛琪（1996—），女，回族，硕士生；电子信箱：liuyuanqi@sjtu.edu.cn。第一联系人：（孙思远、刘媛琪并列第一作者）
基金资助:
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);上海市自然科学基金(21ZR1436900);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)

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

SUN Siyuan¹^,²(), LIU Yuanqi¹^,²(), CUI Yiwen¹^,², HUANG Zihan¹^,², MEI Li²^,³, DAI Qinggang⁴, JIANG Lingyong¹^,²()

^1.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
^2.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
^3.Department of Oral Sciences, Faculty of Dentistry, University of Otago, Dunedin 9016, New Zealand
^4.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

Received:2024-02-01 Accepted:2024-03-21 Online:2024-06-28 Published:2024-06-28
Contact: JIANG Lingyong E-mail:ssy1412832053@163.com;liuyuanqi@sjtu.edu.cn;jianglingyong@sjtu.edu.cn
Supported by:
“Two-Hundred Talents” Program of Shanghai Jiao Tong University School of Medicine(20221809);Fundamental Research Funds for the Central Universities(YG2023ZD14);National Natural Science Foundation of China(82071083);Natural Science Foundation of Shanghai(21ZR1436900);Hainan Provincial Natural Science Foundation(824MS152);Shanghai Science and Technology Innovation Action Plan-International Science and Technology Cooperation Program(23410713600);Cross-Disciplinary Research Fund of Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine(JYJC202116);Biomaterials and Regenerative Medicine Institute Cooperative Research Project, Shanghai Jiao Tong University School of Medicine(2022LHB02);Original Exploration Project of Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine(JYYC003)

摘要/Abstract

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Rosa26^{dnRARα/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 receptor α mutation，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 receptor α，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小鼠模拟VAD所致颅颌面骨畸形，为未来VAD所致颅颌面骨畸形出生后致病机制与潜在靶点的研究提供了新的模型与途径。

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

Abstract:

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 receptor α (dnRARα) mutation were obtained by hybridization through Osx^Cre and Rosa26^{dnRARα/dnRARα} mice, 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 receptor α (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 RARα protein 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^Cre;Rosa26^dn/dn mice compared to their control littermates, and was also inhibited in the Ad-Cre-infected group compared to the Ad-eGFP-infected group (P<0.05). Micro-CT and 3D reconstruction suggested that the skull of 6-week-old Osx^Cre;Rosa26^dn/dn mice exhibited VAD-like craniofacial skeletal deformities, including smaller size of the skull and osteogenesis imperfecta compared to their control littermates. Conclusion ·An osteoblast-specific dnRARα expressing mouse model that can mimic VAD-like craniofacial skeletal deformity is successfully constructed, therefore providing a new model for exploring the pathogenesis and therapeutic targets of VAD-like craniofacial skeletal deformity in the future.

Key words: vitamin A deficiency (VAD), craniofacial skeletal deformity, osteoblast, conditional dominant-negative mutant expressing mice, retinoic acid receptor (RAR)

中图分类号:

孙思远, 刘媛琪, 崔怡雯, 黄紫晗, 梅李, 代庆刚, 江凌勇. 成骨细胞条件性视黄酸信号失活小鼠模型的构建与验证[J]. 上海交通大学学报（医学版）, 2024, 44(6): 676-686.

SUN Siyuan, LIU Yuanqi, CUI Yiwen, HUANG Zihan, MEI Li, DAI Qinggang, JIANG Lingyong. Generation and validation of the conditional osteoblast-specific retinoic acid signaling inhibition mouse model[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024, 44(6): 676-686.

图/表 6

表1 小鼠基因型鉴定的PCR引物

Tab 1 PCR primers for mouse genotyping

Primer name	Forward (5′→3′)	Reverse (5′→3′)
dnRARα-mut	ATGGTGTACACGTGTCACC	CACCTTCTCAATGAGCTCC
dnRARα-wt	CCAAAGTCGCTCTGAGTTGTTATC	GAGCGGGAGAAATGGATATG
OsxCre	TGCCACGACCAAGTGACAGCAATG	ACCAGAGACGGAAATCCATCGCTC

图1 Cre-LoxP系统实现 dnRARα 基因条件性表达的原理示意图Note： A. Schematic diagram of the structure of the dominant negative protein dnRARα. B. Schematic diagram of the Cre-LoxP system in Osx+ cell lineage of OsxCre;Rosa26dn/dn mice. C. Schematic diagram of the activity of retinoic acid signaling in Osx+ cell lineage of OsxCre mice and OsxCre;Rosa26dn/dn mice. AF1—activation function 1; AF2—activation function 2; DBD—DNA binding domain; LBD—ligand binding domain; prom.—promoter.

Fig 1 Schematic diagram of conditional dnRARα gene expressing by Cre-LoxP system

图2 小鼠杂交方案与基因型鉴定Note： A. Schematic diagram of hybridization strategy among mice. B. Genotype identification by tails of mice. N—negative control.

Fig 2 Mouse hybridization and genotyping

图3 OsxCre;Rosa26dn/dn 小鼠成骨细胞视黄酸信号失活效率验证Note： A. The RARα protein level of femur bone BMSCs after osteogenic induction from OsxCre mice and OsxCre;Rosa26dn/dn mice by Western blotting. B. The RARα protein level of parietal bone cells after osteogenic induction from OsxCre mice and OsxCre;Rosa26dn/dn mice by Western blotting. C. The RARE activity of parietal bone cells from OsxCre mice and OsxCre;Rosa26dn/dn mice by dual luciferase gene reporter assay. PGL3-basic is blank plasmid group. OBS—osteoblast; ns—no significant difference.

Fig 3 Verification of the inhibition of osteoblastic retinoic acid signaling in OsxCre;Rosa26dn/dn mice

图4 Cre-LoxP系统体外成骨细胞视黄酸信号失活效率验证Note： A. The RARα protein level of adenovirus-infected femur bone BMSCs after osteogenic induction from Rosa26dn/dn mice detected by Western blotting. B. The RARα protein level of adenovirus-infected parietal bone cells after osteogenic induction from Rosa26dn/dn mice detected by Western blotting. C. The RARE activity of adenovirus-infected parietal bone cells from Rosa26dn/dn mice detected by dual luciferase gene reporter assay. PGL3-basic is blank plasmid group. ns—no significant difference; OBS—osteoblast.

Fig 4 Verification of the inhibition of osteoblastic retinoic acid signaling by Cre-LoxP system in vitro

图5 OsxCre;Rosa26dn/dn 小鼠颅颌面骨畸形分析Note： A. The three-dimensional (3D) reconstruction of the Micro-CT of 6-week-old mouse skulls. B. Bone segmentation of the mouse skulls. C. Analysis of the length of skulls. D. Analysis of the width of skulls. E. Analysis of the length of mandibles. F. The 3D reconstruction of the craniofacial bones. G. Analysis of the volume of craniofacial bones, including mandible, nasal bone, frontal bone, parietal bone, interparietal bone, occipital bone, premaxilla, and maxilla.

Fig 5 Craniofacial skeletal deformity analysis of OsxCre;Rosa26dn/dn mice

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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

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