microRNA-224在牙釉质发育中的作用及机制研究

Enamel, the hardest mineralized tissue in vertebrates, is composed of tightly packed hydroxyapatite crystals and confers the protection and masticatory function in human body. Enamel formation relies on sequentially ameloblast differentiation throughout the dental epithelial sheet on enamel organ, and enamel mineralization orchestrated by mature ameloblast though cellular transportation. During mineralization, there are nearly 8+ protons released into the extracellular matrix accompanied by the formation of every unit of hydroxyapatite crystal, and the pH value decreasing in the extracellular space. This event continues throughout amelogenesis, yet peaks in the maturation phase. However, pH of the enamel extracellular microenvironment in secretory stage remains neutral. Furthermore, pH in the enamel matrix shifts from acidic to neutral periodically in later maturation stage, which indicates a tight modulation of pH homeostasis. As previously reported by us and others, ameloblasts play a critical role in regulating pH through various mechanisms, among which is the buffering system of ion channels expressed in differentiated ameloblasts.It has been proposed that several ion channels were involved in regulation of pH per se during crystal mineralization, including NBCe1 (encoded by SLC4A4), AE2 (encoded by SLC4A2) and CFTR (encoded by CFTR).In these proposed models, ameloblasts were viewed as a polarized layer of cells analogous to other bicarbonate transporting (secretory) epithelia, with anion exchanger AE2 and CFTR at the apical and NBCe1 at the basolateral membrane maintaining and modulating bicarbonate secretion. Same as other developmental events, the expression of these ion channels is driven by changes in gene expression, which is regulated both by internal and external factors modulated in layers both genetically and epigenetically. The spatio-temporal expression of microRNAs has drawn much attention in recent years. It has been indicated in our previous study that microRNA-224 was differentially expressed in different stage of tooh development. Prediction tools indicated miR-224 as candidate to target CFTR and SLC4A4 mRNA. The present study aimed to investigate the potential role of miR-224 as a regulator of ameloblast differentiation and enamel mineralization.

牙釉质是脊椎动物体内最坚硬的矿化组织，由紧密排列的羟基磷灰石晶体组成。其形成过程有赖于终末分化的成釉细胞分泌细胞外釉质基质并介导生物矿化。成釉细胞所表达的离子通道蛋白参与了细胞外基质环境pH值的调节，并推动了牙釉质形成与矿化过程。本课题组前期研究显示，在牙发育过程中存在差异性表达的microRNA miR-224，其预测靶基因包括了成釉细胞缓冲系统中的两种离子通道蛋白基因，SLC4A4及CFTR。miR-224同时对这两种基因的靶向作用提示其可能参与牙釉质发育及矿化过程的调控。本课题试图研究miR-224在人牙胚成釉细胞系细胞中的时空性表达特点，并通过体外细胞学实验验证其与靶基因mRNA的直接作用关系，及miR-224对上皮细胞分化的影响。同时利用动物实验模型，在体内验证miR-224对牙釉质形成与矿化的影响，进一步阐明miR-224在牙釉质发育过程中的作用及分子生物学机制。

牙釉质是脊椎动物体内最坚硬的矿化组织，由紧密排列的羟基磷灰石晶体组成。其形成过程有赖于终末分化的成釉细胞分泌细胞外釉质基质并介导生物矿化。成釉细胞所表达的离子通道蛋白参与了细胞外基质环境pH值的调节，并推动了牙釉质形成与矿化过程。课题前期研究显示，牙发育过程中存在差异性表达的microRNA miR-224，其预测靶基因包括了成釉细胞缓冲系统中的两种离子通道蛋白基因SLC4A4和CFTR。本课题验证了miR-224在人成釉细胞分化不同阶段具有时空性表达特点，随成釉分化过程表达下调，而成釉细胞离子通道蛋白SLC4A4和CFTR基因表达上升，与miR-224的表达趋势呈负相关性。利用miR-224的拟似物和拮抗物模拟细胞内miR-224的过表达和低表达状态，发现miR-224在人成釉细胞及口腔上皮细胞中具有功能性调控SLC4A4和CFTR表达水平的作用，且直接与其mRNA的3’ 非编码区结合，在转录后水平特异性调控离子通道蛋白的表达。利用miR-224的拟似物构建小鼠切牙颈环区miR-224过表达的动物模型，验证了miR-224对小鼠切牙牙釉质发育过程的调节作用，miR-224过表达下调成釉细胞离子通道蛋白SLC4A4和CFTR的表达，影响牙釉质微观结构的形成、钙磷的含量构成比及牙釉质的显微硬度，提示miR-224对牙釉质矿化的调控作用。该研究探索了miR-224通过调控成釉细胞离子通道蛋白SLC4A4和CFTR的表达，调控牙釉质矿化过程，完善microRNA在牙釉质发育过程中的调控作用理论。