牙髓干细胞分化为成牙本质样细胞，形成修复性牙本质，是牙髓损伤修复的核心所在。N6甲基腺嘌呤(m6A)甲基转移酶METTL3介导m6A RNA甲基化在损伤应答、干细胞命运决定中发挥关键作用，但在牙髓干细胞分化中尚无报道。申请人预实验发现，在修复性牙本质形成区的牙髓组织中METTL3表达上调，且在牙髓干细胞成牙本质向分化过程中METTL3介导m6A修饰水平升高。RNA-seq进一步筛选METTL3的靶基因，SOX9表达变化显著并具有相关性。那么，METTL3如何调控牙髓干细胞分化，是否与SOX9的m6A修饰有关？本课题拟构建METTL3抑制、过表达和酶活性突变的牙髓干细胞，探讨METTL3对成牙本质向分化进程的调控作用，以m6A免疫共沉淀分析METTL3靶向修饰SOX9的机制，并结合体内模型进行验证。本研究有助于揭示牙髓干细胞分化过程中的RNA表观遗传学机制，为活髓保存提供新的思路和潜在靶点。

The odontogenic differentiation of dental pulp stem cells is a key program in the study of restorative dentin formation and regeneration. N6 methyladenine (m6A) methyltransferase METTL3-mediated m6A RNA methylation plays a key role in injury response and stem cell fate determination, but it has not been reported in the differentiation of dental pulp stem cells. In the preliminary experiment, we found METTL3 expression was up-regulated in the pulp tissue of the restorative dentin formation area, and METTL3-mediated m6A modification was induced during odontogenic differentiation of pulp stem cells. We further explored the target genes of METTL3 by RNA-seq, the expression of SOX9 was significantly changed. So, how does METTL3 regulate the differentiation of dental pulp stem cells? Is it related to the m6A modification of SOX9? In this study, we intend to construct dental pulp stem cells transfected with METTL3 inhibition, over-expression and enzyme activity mutation lentivirus vector to investigate the regulatory mechanism of METTL3 on odontogenic differentiation at gene and protein levels. We use m6A co-immunoprecipitation to investigate that SOX9 was modified by METTL3 and verify it in vivo model. This study is helpful to reveal the mechanism of RNA epigenetics during the differentiation of dental pulp stem cells, and provide new ideas and potential targets for the preservation of living pulp.