NRAGE在牙髓细胞成牙本质向分化中的作用及机制研究

Stimulated by exogenous stimulations (such as caries, erosion, etc.), pulpo-dentinal complex will induce dental pulp cells differentiate into odontoblasts which results in reparative dentine formation. Neurotrophin receptor-interacting melanoma antigen-encoding gene homology (NRAGE) is related to the growth and cell differentiation. Our previous study confirmed that NRAGE is a potent regulator of proliferation and odontoblastic differentiaioin of mDPCs, which might through the NF-κB signaling pathway. Meanwhile, knockdown of NRAGE in mDPCs enhanced the mRNA level of autophagy related gene. Autophagy inhibitor regulated the effect of NRAGE on odontoblastic differentiation of mDPCs. we hypothesized that: "NRAGE regulates the odontoblastic differentiation of mDPCs through autophage which depend on NF-κB signaling pathways". We will analysis the molecular mechanism of NRAGE on odontoblastic differentiation of DPCs in vivo and in vitro:1) Test the effects of NRAGE on the dentin development in mice;2) Whether adjusting the expression level of NRAGE could change the expression of autophagy related gene and NF-κB;3) Regulating NF-κB signaling pathway and/or autophagy, to explain the role of NRAGE/NF-κB/autophagy in odontoblastic differentiaion. Base on above works, we will try to explain the role and molecular mechanism of NRAGE in dentin development and odontoblastic differentiation.

牙髓牙本质复合体受到外源性刺激时，牙髓细胞向成牙本质细胞分化，诱导修复性牙本质的形成。神经生长因子受体作用的黑色素瘤抗原编码基因同源蛋白（NRAGE)与生长发育和细胞分化密切相关。我们前期研究证实低表达NRAGE可能激活NF-kB信号通路促进牙髓细胞向成牙本质细胞分化。进一步研究发现，NRAGE低表达上调牙髓细胞中自噬相关基因的表达。我们提出科学假说：“NRAGE通过NF-κB信号通路调控自噬，进而调控牙髓细胞成牙本质分化”。为了阐述NRAGE在牙髓细胞成牙本质分化中的分子机制，我们将从体内和体外两个角度分析：1）NRAGE对小鼠牙本质发育的影响；2）调节NRAGE的表达是否影响NF-kB信号分子和自噬相关基因的表达；3）探索NRAGE/NF-κB/自噬在小鼠牙本质发育及牙髓细胞成牙本质分化中的机制。通过上述研究，阐述NRAGE在牙髓细胞成牙本质向分化及牙本质发育中的重要作用及机制。

牙髓牙本质复合体受到外源性刺激时，牙髓细胞向成牙本质细胞分化，诱导修复性牙本质的形成。神经生长因子受体作用的黑色素瘤抗原编码基因同源蛋白（NRAGE)与生长发育和细胞分化密切相关。基于本课题组前期研究：NRAGE可能通过NF-κB信号通路调节牙髓细胞的成牙本质向分化，且NRAGE调控自噬相关基因的表达。本项目拟将通过体内和体外实验，阐述NRAGE在牙髓细胞成牙本质向分化中的作用及分子机制。研究内容主要包括：NRAGE对牙釉质发育的影响；NRAG成牙本质细胞系的成牙本质向分化的作用及分子调控机制；NRAGE对牙釉质调控的分子机制。研究发现NRAGE敲除小鼠牙釉质发育异常；发现NRAGE是与NF-kB调节复合物IkB中的亚型IKKβ结合，从而阻碍了IKKβ与NF-kB的结合，促进了NF-kB的活化，活化的NF-kB入核后作为转录因子，激活了p65的磷酸化，从而促进了牙髓细胞，成牙本质细胞向牙釉质细胞的分化；在成牙釉质细胞系（ALC细胞系）中敲减NRAGE基因，发现ALC细胞被阻滞于G2/M期，细胞的凋亡减少，自噬被激活，体内也发现敲除NRAGE后自噬相关基因（ATG5和ATG7）在小鼠的成釉细胞中高表达，提示NRAGE调控牙釉质发育与自噬相关。本研究有望阐明牙釉质发育的新机制，确定NRAGE可作为AI的诊断和干预靶点。