【作者】 杜玮； 杜文； 郑黎薇； 周学东；

【机构】 口腔疾病防治全国重点实验室国家口腔医学中心国家口腔疾病临床医学研究中心； 四川大学华西口腔医院牙体牙髓病科； 四川大学华西口腔医院口腔修复Ⅱ科； 四川大学华西口腔医院儿童口腔科；

【摘要】 目的 :牙发育是一个连续、复杂的生物学过程,有赖于外胚层来源的上皮细胞和其覆盖的颅神经嵴来源的间充质细胞相互作用、共同分化,任何阶段受到干扰均可能导致牙发育异常。对牙发育的研究长期以来主要侧重于各种化学因子及受体介导的信号通路。近年来,机械应力在牙发育过程中的重要性日益受到关注,本研究旨在探究重要的应力相关因子Myosin II在牙发育过程中的作用及机制,完善对牙发育过程的全面认识。材料与方法:利用Myosin II编码基因Myh9、Myh10构建牙上皮条件性敲除小鼠(K14CreER;Myh9fl/fl,Myh10fl/fl双敲小鼠,即Myh9/10epi-cko小鼠),孕鼠于E10.5时他莫昔芬灌胃,收集E12.5-E15.5不同牙发育时期的小鼠切牙,通过3D重建、免疫荧光染色观察敲除Myh9/10后切牙形态及下游信号表达变化;利用活细胞成像检测切牙牙上皮细胞运动状态。同时,给予6-8周成年小鼠他莫昔芬,H&E染色及免疫荧光染色观察小鼠切牙形态变化及下游信号表达变化。结果:在E12.5小鼠下颌组织中加入Myosin II抑制剂Blebbistatin进行体外培养后,切牙的牙胚形成明显受阻。Myh9/10epi-cko小鼠切牙早期形态、内陷程度及体积均较对照组偏小,成釉器颈部无法正常缩窄,甚至出现空泡囊腔。而条件敲除Myosin II后,小鼠切牙laCL的细胞增殖平衡状态改变,伴随组织形态异常,成釉分化推迟。活细胞成像(live imaging)发现敲除Myosin II后,小鼠切牙细胞运动紊乱,无法正常与临近细胞相互作用。免疫荧光染色发现,细胞黏附连接相关标记物β-catenin以及特定胞外区E-cadherin的表达在敲除Myosin II后明显下降。结论:Myosin II通过维持正确的细胞黏附连接及细胞运动状态,促进正常牙发育进程。更多还原

【Abstract】 Objectives: Tooth development requires epithelial invagination into the mesenchyme and subsequent formation of correct shapes. While signaling ligands are critical for tooth development, how cells respond to morphogens and become self-organized to enable tissue shape changes remains an open question. Here we use the mouse dentition as a model to investigate how non-muscle myosin Ⅱ participate in propel epithelial invagination and later tooth development.Methods: We first established the spatiotemporal expression of myosin Ⅱ components in the developing mouse incisor. To investigate the functional role of myosin Ⅱ, we conditionally deleted myosin heavy chains ⅡA and ⅡB in dental epithelial cells and assessed tissue and cellular phenotypes using histological analysis, immunohistochemistry and live imaging.Results: We found that both Myosin ⅡA and ⅡB are expressed in the incisor epithelium and required for proper tooth development and amelogenesis. Timelapse microscopy revealed that myosin Ⅱ is essential for efficient and persistent convergent cell movement in the suprabasal layer to facilitate invagination. Furthermore, myosin Ⅱ is essential for strong cell-cell adhesion.Conclusion: These findings demonstrate that myosin Ⅱ drives cell-cell adhesion and convergent cell movement to enable effective dental epithelial invagination and amelogenesis.更多还原