新型bHLH转录因子CartD调控小鼠前成釉细胞迁移和分化的研究

Ameloblast secretes enamel matrix proteins which mineralize to form the dental enamel that is necessary for daily chewing. The maturation of ameloblast is a stepwise process in which the Sox2 positive dental epithelial stem cell (DESCs) transit into the terminally differentiated ameloblast. Currently the regulatory network of ameloblast maturation is still not fully understood. In our previous studies, we screened a rat tooth germ cDNA library using the yeast two-hybrid (Y2H) system to clone bHLH proteins. One of the clones, which we named as CartD, was identified as a novel class B bHLH transcription factor. CartD mRNA was strongly expressed in developing mouse molars and incisors. In situ hybridization and immunostaining showed that CartD localized in the inner dental epithelium of mouse molars, it is highly expressed in the pre-ameloblast but relatively lower expression in the DESCs and terminally differentiated ameloblast. In the cell culture system, overexpression of CartD both in the pre-ameloblast and MDCK cells induces the epithelium mesenchyme transiton (EMT), which promotes the cell migration. Based on those results, we proposed that CartD may have important roles in regulating the amleogeneis process. In order to elucidate the role of CartD in mouse tooth development, our next step work will focus on the following three questions: 1, the regulatory mechanism of CartD during the transition from DESCs to pre-amelobast; 2, the signaling cascade of CartD mediated EMT and cell migration; the relationship between ameloblast terminal differentiation and CartD. We are proposing to use RNA-seq to analyze the whole transcriptome gene expression changes caused by CartD overexpression or knockdown; we will use ChIP-seq to identify the target genes which are directly regulated by CartD; Confocal microscopy and Time lapse microscopy will be used to screen the EMT related dynamic cell morphological changes and migration which are induded by CartD. Our study will provide a comprehensive understanding of CartD regulated ameloblast maturation process, which can be used as theoretical foundation for the tooth regeneration.

本课题前期研究中采用酵母双杂交 (Y2H) 发现了特异性表达于内釉上皮的bHLH转录因子并命名为CartD。CartD高表达于前成釉细胞中，在牙源性上皮干细胞以及终端分化的成釉细胞中表达量较低。体外实验发现CartD能诱导EMT，促进细胞迁移。本研究以此为基础，将研究以下问题：1，牙源性上皮干细胞到前成釉细胞的细胞命运的转变过程中CartD的调控作用；2， CartD通过介导EMT促进细胞迁移的调控原理；3，成釉细胞终端分化中CartD发挥的调控作用。拟采用的研究手段包括：CartD过表达以及低表达细胞系的建立；采用RNA-seq在转录组水平进行基因表达分析； ChIP-seq分析CartD直接调节的目的基因；激光共聚焦显微镜和Time lapse显微镜观察由CartD介导的 EMT以及细胞迁移。通过以上研究，阐明CartD在成釉细胞成熟过程中的调节作用，完善成釉细胞分化的分子机制。

bHLH家族转录因子在调控肌肉细胞分化，骨细胞分化，神经发育中发挥着重要作用。目前，并无BHLH转录因子调控小鼠牙齿发育的研究。在在本课题中，我们采用酵母双杂交技术，从大鼠牙胚cDNA文库中克隆新型的bHLH转录因子，我们将该转录因子命名为AmeloD。AmeloD在基因水平和蛋白水平均特异性表达于小鼠前成釉细胞中，而在小鼠牙源性上皮干细胞以及终端分化的成釉细胞中午表达。为了鉴定AmeloD蛋白在小鼠牙发育过程中得作用，我们进行了系统的体内以及体外研究。我们第一次构建了AmeloD基因敲除小鼠，以及AmeloD和EPFN蛋白双敲除小鼠。通过系统研究结果，我们发现AmeloD蛋白主要调控前成釉细胞的细胞迁移能力。敲除AmeloD蛋白之后，小鼠表现出多种牙发育障碍，包括釉质发育不全。我们的研究证明，AmeloD是调控小鼠牙齿发育的重要调节因子。