E3泛素连接酶March6抑制成牙本质细胞分化以及牙本质生成

doi:10.13701/j.cnki.kqyxyj.2025.08.004

口腔医学研究 ›› 2025, Vol. 41 ›› Issue (8): 657-663.DOI: 10.13701/j.cnki.kqyxyj.2025.08.004

E3泛素连接酶March6抑制成牙本质细胞分化以及牙本质生成

冯豪, 袁国华^*

口颌系统重建与再生全国重点实验室,口腔生物医学教育部重点实验室,口腔医学湖北省重点实验室,武汉大学口腔医学院湖北武汉 430079

收稿日期:2025-04-10 出版日期:2025-08-28 发布日期:2025-08-15
通讯作者: ^*袁国华,E-mail:yuanguohua@whu.edu.cn
作者简介:冯豪(1999~ ),男,河南周口人,硕士在读,研究方向:牙齿发育生物学。
基金资助:
国家自然科学基金(编号:82370914)

E3 Ubiquitin Ligase March6 Suppresses Odontoblast Differentiation and Dentinogenesis

FENG Hao, YUAN Guohua^*

State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Key Laboratory of Oral Biomedicine Ministry of Education, Hubei Key Laboratory of Stomatology, School & Hospital of Stomatology, Wuhan University, Wuhan 430079, China

Received:2025-04-10 Online:2025-08-28 Published:2025-08-15

摘要/Abstract

摘要： 目的: 明确膜相关的RING-CH型E3泛素连接酶6(membrane-associated RING finger protein 6, March6)在成牙本质细胞分化以及牙本质生成中的作用。方法: 采用免疫组织化学染色(immunohistochemistry, IHC)分析不同发育阶段小鼠牙齿切片,分析March6的时空表达特征。体外敲低小鼠牙乳头细胞(mouse dental papilla cells, mDPCs)中的March6,并进行矿化诱导,分别使用实时定量PCR、Western bolt、碱性磷酸酶(alkaline phosphatase, ALP)染色以及茜素红染色,评估March6对mDPCs成牙本质细胞向分化潜能的影响。将Prx1^cre与March6^flox/flox小鼠配繁,获得牙齿间充质中特异性敲除March6的小鼠(March6 cKO, Prx1^cre;March6^flox/flox)。通过IHC验证March6被特异性敲除。随后,对出生后10 d的March6 cKO及其同窝对照小鼠的下颌行切片苏木素-伊红染色、显微CT(micro-computed tomography, Micro-CT)观察,以确定March6在体内对牙本质生成的影响。结果: March6在胚胎时期小鼠牙齿间充质中表达水平较低,而在成牙本质细胞形成后,March6在成牙本质细胞中高表达。体外敲低mDPCs中的March6,并诱导其向成牙本质细胞分化,牙本质基质蛋白1(dentin matrix protein 1,Dmp1)和牙本质涎磷蛋白(dentin sialophosphoprotein,Dspp)的mRNA以及蛋白水平相较于对照显著上调。同时,细胞ALP活性和钙化结节生成能力在敲低组中也明显增强。组织学形态观察以及Micro-CT分析结果显示,March6 cKO小鼠的牙本质层相较于同窝对照明显增厚。结论: E3泛素连接酶March6对成牙本质细胞分化以及牙本质生成发挥抑制作用。

关键词: March6, 成牙本质细胞分化, 牙本质生成, 基因敲除

Abstract: Objective: To investigate the role of E3 ubiquitin ligase March6 in odontoblast differentiation and dentinogenesis. Methods: Immunohistochemistry (IHC) staining was performed on mouse tooth sections at different developmental stages to determine the spatiotemporal expression pattern of March6. March6 was knocked down in mouse dental papilla cells (mDPCs) in vitro, followed by mineralization induction. Quantitative real-time PCR, western blot, alkaline phosphatase (ALP) staining, and alizarin red S staining were subsequently performed to evaluate the effect of March6 on the odontoblastic differentiation potential of mDPCs. Prx1^cre mice were crossed with March6^flox/flox mice to generate conditional knockout (cKO) mice with mesenchyme-specific deletion of March6 in the dental tissue (March6 cKO, Prx1^cre;March6^flox/flox). Specific deletion of March6 was validated by IHC. Mandibles from postnatal day 10 (PN10) March6 cKO and littermate control mice were analyzed by hematoxylin and eosin staining and micro-computed tomography (micro-CT) to evaluate the in vivo effect of March6 on dentinogenesis. Results: March6 exhibited low expression in the dental mesenchyme of embryonic mice, while high expression was observed in odontoblasts after their differentiation. Knockdown of March6 in mDPCs significantly upregulated the mRNA and protein levels of Dmp1 and Dspp during odontoblastic differentiation. Furthermore, ALP activity and calcified nodule formation were markedly increased in March6 deficient cells. Histological analysis and micro-CT imaging revealed a significantly thicker dentin layer in March6 cKO mice compared with littermate controls. Conclusion: The E3 ubiquitin ligase March6 negatively regulates odontoblast differentiation and dentinogenesis.

Key words: March6, odontoblast differentiation, dentinogenesis, gene knockout

冯豪, 袁国华. E3泛素连接酶March6抑制成牙本质细胞分化以及牙本质生成[J]. 口腔医学研究, 2025, 41(8): 657-663.

FENG Hao, YUAN Guohua. E3 Ubiquitin Ligase March6 Suppresses Odontoblast Differentiation and Dentinogenesis[J]. Journal of Oral Science Research, 2025, 41(8): 657-663.

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E3泛素连接酶March6抑制成牙本质细胞分化以及牙本质生成

E3 Ubiquitin Ligase March6 Suppresses Odontoblast Differentiation and Dentinogenesis

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