上海交通大学学报（医学版）

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2024 , Vol. 44 >Issue 6: 676 - 686

DOI: https://doi.org/10.3969/j.issn.1674-8115.2024.06.002

牙颌面畸形专题

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 ,
  • 刘媛琪 ,
  • 崔怡雯 ,
  • 黄紫晗 ,
  • 梅李 ,
  • 代庆刚 ,
  • 江凌勇
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  • 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
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）
江凌勇，电子信箱：jianglingyong@sjtu.edu.cn。

收稿日期: 2024-02-01

  录用日期: 2024-03-21

  网络出版日期: 2024-06-28

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)

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

  • Siyuan SUN ,
  • Yuanqi LIU ,
  • Yiwen CUI ,
  • Zihan HUANG ,
  • Li MEI ,
  • Qinggang DAI ,
  • Lingyong JIANG
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  • 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
JIANG Lingyong, E-mail: jianglingyong@sjtu.edu.cn.

Received date: 2024-02-01

  Accepted date: 2024-03-21

  Online published: 2024-06-28

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)

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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OsxCre 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 receptor α mutation，dnRARα）条件性过表达小鼠OsxCre;Rosa26dn/dn，实现成骨细胞视黄酸信号条件性失活以模拟VAD状态。取OsxCre;Rosa26dn/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OsxCre;Rosa26dn/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Rosa26dn/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OsxCre;Rosa26dn/dn 模型小鼠颅颌面骨畸形表型。结果·Western blotting结果显示，OsxCre;Rosa26dn/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OsxCre;Rosa26dn/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Rosa26dn/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OsxCre;Rosa26dn/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

本文引用格式

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 . DOI: 10.3969/j.issn.1674-8115.2024.06.002

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 OsxCre;Rosa26dn/dn mice expressing osteoblast-specific dominant-negative retinoid acid receptor α (dnRARα) mutation were obtained by hybridization through OsxCre and Rosa26dnRARα/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 OsxCre;Rosa26dn/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 OsxCre;Rosa26dn/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 Rosa26dn/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 OsxCre;Rosa26dn/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 OsxCre;Rosa26dn/dn mice compared to that of their control littermates, and also increased in the Ad-Cre-infected femur and parietal osteoblasts of Rosa26dn/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 OsxCre;Rosa26dn/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 OsxCre;Rosa26dn/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)

参考文献

1 TIERNEY M T, POLAK L, YANG Y H, et al. Vitamin A resolves lineage plasticity to orchestrate stem cell lineage choices[J]. Science, 2024, 383(6687): eadi7342.
2 SEUFERT J, KRISHNAN N, DARMSTADT G L, et al. Subnational estimates of vitamin A supplementation coverage in children: a geospatial analysis of 45 low- and middle-income countries[J]. Public Health, 2024, 228: 194-199.
3 O'CONNOR E A, EVANS C V, IVLEV I, et al. Vitamin and mineral supplements for the primary prevention of cardiovascular disease and cancer: updated evidence report and systematic review for the US preventive services task force[J]. JAMA, 2022, 327(23): 2334-2347.
4 CHAKRABORTY U, CHANDRA A. Bitot's spots, dry eyes, and night blindness indicate vitamin A deficiency[J]. Lancet, 2021, 397(10270): e2.
5 SHEFTEL J, VAN STUIJVENBERG M E, DHANSAY M A, et al. Chronic and acute hypervitaminosis A are associated with suboptimal anthropometric measurements in a cohort of South African preschool children[J]. Am J Clin Nutr, 2022, 115(4): 1059-1068.
6 GHYSELINCK N B, DUESTER G. Retinoic acid signaling pathways[J]. Development, 2019, 146(13): dev167502.
7 LOHNES D, MARK M, MENDELSOHN C, et al. Function of the retinoic acid receptors (RARs) during development (Ⅰ). Craniofacial and skeletal abnormalities in RAR double mutants[J]. Development, 1994, 120(10): 2723-2748.
8 ONO K, SANDELL L L, TRAINOR P A, et al. Retinoic acid synthesis and autoregulation mediate zonal patterning of vestibular organs and inner ear morphogenesis[J]. Development, 2020, 147(15): dev192070.
9 ABADIE R B, STAPLES A A, LAUCK L V, et al. Vitamin A-mediated birth defects: a narrative review[J]. Cureus, 2023, 15(12): e50513.
10 DAMM K, HEYMAN R A, UMESONO K, et al. Functional inhibition of retinoic acid response by dominant negative retinoic acid receptor mutants[J]. Proc Natl Acad Sci U S A, 1993, 90(7): 2989-2993.
11 CUNNINGHAM C J, CHOI R B, BULLOCK W A, et al. Perspective: the current state of Cre driver mouse lines in skeletal research—challenges and opportunities[J]. Bone, 2023, 170: 116719.
12 GEMBERLING M P, SIKLENKA K, RODRIGUEZ E, et al. Transgenic mice for in vivo epigenome editing with CRISPR-based systems[J]. Nat Methods, 2021, 18(8): 965-974.
13 ROSSELOT C, SPRAGGON L, CHIA I, et al. Non-cell-autonomous retinoid signaling is crucial for renal development[J]. Development, 2010, 137(2): 283-292.
14 DAI Q, SUN S, JIN A, et al. Osteoblastic RAR inhibition causes VAD-like craniofacial skeletal deformity[J]. J Dent Res, 2023, 102(6): 667-677.
15 VICTORA C G, CHRISTIAN P, VIDALETTI L P, et al. Revisiting maternal and child undernutrition in low-income and middle-income countries: variable progress towards an unfinished agenda[J]. Lancet, 2021, 397(10282): 1388-1399.
16 NUREDIN A, MELIS T, ABDU A O. Clinical vitamin A deficiency among preschool aged children in Southwest Ethiopia[J]. Front Nutr, 2024, 11: 1267979.
17 SCHMIDT M J, STEENKAMP G, CALDWELL P, et al. Radiographic analysis of the thickness of the cranial bones in captive compared to wild-living cheetahs and in cheetahs with hypovitaminosis A[J]. PLoS One, 2021, 16(8): e0255924.
18 WANG F P, TANG Y W, CAI Y J, et al. Intrafollicular retinoic acid signaling is important for luteinizing hormone-induced oocyte meiotic resumption[J]. Genes, 2023, 14(4): 946.
19 BAYBUTT R C, STANDARD J T, DIM D, et al. Cod liver oil, but not retinoic acid, treatment restores bone thickness in a vitamin A-deficient rat[J]. Nutrients, 2022, 14(3): 486.
20 PETRELLI B, BENDELAC L, HICKS G G, et al. Insights into retinoic acid deficiency and the induction of craniofacial malformations and microcephaly in fetal alcohol spectrum disorder[J]. Genesis, 2019, 57(1): e23278.
21 FIRSCHEIN H E. Collagen and mineral accretion rates in bone during vitamin A deficiency[J]. Am J Physiol, 1970, 219(5): 1183-1187.
22 KNAPIK J J, HOEDEBECKE S S. Vitamin A and bone fractures: systematic review and meta-analysis[J]. J Spec Oper Med, 2021, 21(2): 100-107.
23 TIMBERLAKE A T, MCGEE S, ALLINGTON G, et al. De novo variants implicate chromatin modification, transcriptional regulation, and retinoic acid signaling in syndromic craniosynostosis[J]. Am J Hum Genet, 2023, 110(5): 846-862.
24 LAMMER E J, CHEN D T, HOAR R M, et al. Retinoic acid embryopathy[J]. N Engl J Med, 1985, 313(14): 837-841.
25 WU Y, KUROSAKA H, WANG Q, et al. Retinoic acid deficiency underlies the etiology of midfacial defects[J]. J Dent Res, 2022, 101(6): 686-694.
26 LOKKEN-TOYLI K L, DIAZ-OCHOA V E, CAMACHO L, et al. Vitamin A deficiency impairs neutrophil-mediated control of Salmonella via SLC11A1 in mice[J]. Nat Microbiol, 2024, 9(3): 727-736.
27 YU H R, WU J, LI K X, et al. Integrated analysis of murine cornea identifies JAK/STAT signaling pathway upregulated specifically in female vitamin A deficient mice[J]. Exp Eye Res, 2023, 237: 109714.
28 RADHAKRISHNAN R, LEUNG M, ROEHRICH H, et al. Mice lacking the systemic vitamin A receptor RBPR2 show decreased ocular retinoids and loss of visual function[J]. Nutrients, 2022, 14(12): 2371.
29 CHIU M, WATSON S. Xerophthalmia and vitamin A deficiency in an autistic child with a restricted diet[J]. BMJ Case Rep, 2015, 2015: bcr2015209413.
30 CHEN J Q, SHI Y, REGAN J, et al. Osx-Cre targets multiple cell types besides osteoblast lineage in postnatal mice[J]. PLoS One, 2014, 9(1): e85161.
31 BOK S, YALLOWITZ A R, SUN J, et al. A multi-stem cell basis for craniosynostosis and calvarial mineralization[J]. Nature, 2023, 621(7980): 804-812.
32 YEE M M F, CHIN K Y, IMA-NIRWANA S, et al. Vitamin A and bone health: a review on current evidence[J]. Molecules, 2021, 26(6): 1757.
33 LI Q W, XU R S, LEI K X, et al. Insights into skeletal stem cells[J]. Bone Res, 2022, 10(1): 61.
34 SUN J, HU L L, BOK S, et al. A vertebral skeletal stem cell lineage driving metastasis[J]. Nature, 2023, 621(7979): 602-609.
35 COLLINS M T, MARCUCCI G, ANDERS H J, et al. Skeletal and extraskeletal disorders of biomineralization[J]. Nat Rev Endocrinol, 2022, 18(8): 473-489.
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