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

项目名称： 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

项目编号： No.81500811

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 医药、卫生

项目作者： 贺兵

作者单位： 四川大学

项目金额： 18万元

中文摘要： 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。

中文关键词： bHLH；转录因子；成釉细胞；细胞迁移；细胞分化

英文摘要： 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.

英文关键词： bHLH transcription factor;ameloblast;cell migration;cell differentiation