基于ME2介导的褪黑素促进细胞有氧代谢调控DPCs成牙本质向分化的分子机制

Accumulating evidence has revealed that melatonin (MT) might be involved in the development of dentine and tooth formation, but the underlying mechanism remains uncertain. Our previous study showed that MT promoted the differentiation and mineralization of rat dental papilla cells (DPCs), accompanied by mitochondrial activity promotion (J Pineal Res.2013, online, IF=7.2). We also found that mitochondrial function was elevated during the process of dentinogenesis, whereas inhibition of mitochondrial respiratory chain complex I could hamper the odontogenic differentiation of DPCs. Further experiments indicated that MT increased malic enzyme 2 (ME2) expression during the odontogenic differentiation, and ME2 is a kind of oxidative carboxylases regulating tricarboxylic acid cycle (TCA cycle), thus promoting ATP production and mitochondrial function. Does MT upregulate TCA cycle, ATP content and oxidative metabolism mediated by ME2, thus promote the odontogenic differentiation of DPCs? This project aimed at exploring the temporal-spatial expression of ME2 in tooth development. Then knockdowning and overexpressing ME2 using the viral plasmid vector will be used to study the effects of MT on the correlation factors of TCA cycle, mitochondrial respiratory chain complex and cellular oxidative metabolism during the odontogenic differentiation of DPCs. We expect to clarify the mechanisms of melatonin promoting the odontogenic differentiation of DPCs via upregulating cellular oxidative metabolism mediated by ME2. This study will provide useful targets for tooth development and regeneration, and it will lay foundation for the study of MT regulating organ development and regeneration.

研究显示褪黑素(MT)参与调控牙齿发育，但机制不明。我们前期研究发现，MT促进牙乳头细胞(DPCs)分化和矿化，并促进DPCs线粒体功能(J Pineal Res，IF=7.2)。我们还发现，DPCs分化过程中线粒体功能明显增强，而抑制线粒体呼吸链复合体Ⅰ活性则抑制其分化；进一步实验发现，MT上调DPCs的苹果酸酶2(ME2)，而ME2可上调细胞三羧酸循环(TCAc)，促进线粒体产生ATP。那么MT是否通过上调ME2促进DPCs线粒体TCAc产生ATP，提高细胞有氧代谢率，达到促DPCs分化作用呢？本项目在研究ME2牙胚发育过程中的表达基础上，分别沉默和过表达ME2的基因，研究MT对DPCs分化过程中TCAc相关因子、呼吸链复合体和有氧代谢率等的影响。以期阐明MT这种促分化效应的有氧代谢供能机制及ME2作用位点，有望为调控牙齿发育和再生提供新靶点，为研究MT调控器官发育和再生机制奠定基础。

牙齿发育过程复杂，受昼夜节律控制。褪黑素（MT）作为调节昼夜节律的主要激素，其调控牙齿发育的潜在机制尚未明确。本研究在检测成牙本质向分化过程中线粒体动力学变化的基础上，探讨褪黑素是否参与牙齿发育的调节及其潜在机制，提示MT可通过增强OPA1介导的线粒体融合促进牙乳头细胞（DPCs）的成牙本质向分化。结合基因测序及体内外实验，本研究还发现，苹果酸酶2（ME2）作为MT的关键下游靶因子，协同调控成牙分化、线粒体融合和呼吸功能。本研究为MT调控牙齿发育机制的研究奠定基础，为牙再生提供了新靶点。本项目各项研究内容进展顺利，并已取得多项成果，本项目资助的研究内容发表SCI期刊研究型论文15篇（其中一篇于12-Jan-2023被J PINEAL RES接收），SCI综述型论文10篇，病例报道1篇，牙发育研究方向的学位论文10篇，其中博士学位论文2篇。项目执行期间共计培养博士后3名和牙发育研究方向的硕博研究生10名，其他方向的硕士研究生4名。