LncRNA DANCR介导Wnt信号通路调控牙髓细胞损伤修复的机制研究

Dental pulp can induce reparative response through odontoblast differentiation of human dental pulp cells in response to external stimuli. In the preliminary studies, we identified DANCR, an anti-differentiation non-coding RNA, exerted important effect on the differentiation of dental pulp cells into odontoblast-like cells, however, the specific mechanism is poorly understood. The Wnt signaling pathway is crucial to odontoblast differentiation. Notably, recent studies have found that NDRG1 can competitively bind to GSK-3β to allow β-catenin to escape degradation, with consequent elevated downstream genes of Wnt signaling.Importantly,we provided evidence that DANCR bound with the NDRG1 by RNA pull-down experiment. These background has led us to the speculation that DANCR may regulate Wnt signal pathway through combining with NDRG1, then inhibit odontoblast differentiation of dental pulp cells to participate in the dental pulp repair. In this study, we attempt to identify whether lncRNA DANCR participate in the regulation of dental pulp cell differentiation. In addition, we will analyse the combination zone of DANCR and NDRG1 and further discuss their interaction mechanism. Furthermore, our study will explore the effects of DANCR mediating function silence of NDRG1 on Wnt signal pathway and odontoblast differentiation of human dental pulp cells, providing insight toward a better understanding of lncRNA DANCR in dental pulp reparative reaction. Knowledge gained in these aspects will set the bases for further research on the reparative response mechanism of dental pulp cells.

牙髓细胞成牙本质向分化是牙髓受外界刺激时作出的防御修复反应。LncRNA DANCR是一种分化抑制基因，课题组前期研究发现其在牙髓细胞成牙本质向分化中发挥作用，但具体机制未明。Wnt信号通路参与调控牙髓细胞分化，新近研究发现，NDRG1竞争性结合该通路的关键分子GSK3β发挥正向调控作用；我们预实验初步证实DANCR与NDRG1相结合。据此我们提出DANCR可能通过沉默NDRG1负向调控Wnt信号通路，抑制牙髓细胞成牙本质向分化从而参与调控牙髓损伤修复的新假设。本研究拟在前期工作基础上，明确LncRNA DANCR在牙髓细胞成牙本质向分化中的具体调控作用；分析DANCR与NDRG1的结合区域及两者相互作用方式；观察DANCR介导的NDRG1沉默对Wnt信号通路及牙髓细胞成牙本质向分化的影响；阐明LncRNA DANCR在牙髓损伤修复中的作用机制，为深入研究牙髓组织防御修复机制提供新思路。

牙源性干细胞具有自我更新和多向分化潜能、获取容易、来源广泛，在再生医学领域具有重要作用。本研究主要研究了长链非编码RNA通过调控牙髓细胞分化进而参与牙髓组织损伤修复再生，同时也进一步拓展研究了牙髓干细胞在牙髓组织再生、牙周组织缺损修复中的作用。获得结果如下：① 为了探讨lncRNA DANCR在hDPCs 成牙本质向分化中的作用。通过功能缺失实验，我们发现低表达DANCR 抑制hDPCs 成牙本质向分化。生物信息分析、荧光素酶报告及RNA免疫沉淀证实lncRNA DANCR与miR-216a结合，且表达负相关。功能实验证实lncRNA DANCR通过直接结合抑制miR-216a发挥对hDPCs分化的调控作用。相关结果提示lncRNA DANCR可作为修复牙髓损伤的潜在治疗靶点。② 进一步揭示了正向调控因子LncRNA CALB2与miR-30b-3p竞争性调节促进hDPSCs成牙本质向分化；③为了探究牙髓干细胞在牙周组织修复中的作用。我们发现DPSCs可通过分泌效应分子诱导巨噬细胞向抗炎的 M2 型转变，进而降低炎症促进牙周组织的损伤修复；④进一步探讨DPSCs如何发挥旁分泌效应，我们发现 hDPSCs外泌体调控成骨和成血管促进组织再生；⑤ 最后，我们初步探讨DPSCs结合牙髓组织来源的脱细胞水凝胶在牙髓再生中的作用, 结果提示牙髓组织脱细胞水凝胶可促进DPSCs粘附、迁移、增殖、成牙本质向分化及成神经向分化，并能促进HUVECs的成管。相关结果探讨了牙髓干细胞作为牙髓-牙周的损伤修复和再生种子细胞的可行性，为口颌面损伤修复机制的研究提供新的视角。