DLK1介导Notch信号通路调控牙胚间充质细胞的成牙诱导潜能的机制研究

Tooth loss is a major problem of oral health. Stem cell-based whole tooth bio-engineering is a promising alternative treatment to the traditional artificial teeth, where cell expansion is required due to the limited number of odontogenic cells that can be obtained in clinic. However, the odontogenic induction capacity of the cells is lost after in vitro culture, which becomes the greatest bottleneck of the research on tooth regeneration in a clinical scenario. We have demonstrated that cultured embryonic dental mesenchymal cells (eDMCs) can be rescued from failure of tooth induction by the community effect created through mixing with fresh eDMCs. However, the mechanism of this effect is still unclear. In pre-experiments, RNA-seq data analysis reveals that Dlk1 was upregulated in the cultured eDMCs with restored odontogenic inductivity; DLK1 treatment can upregulate odontogenic genes Pax9 and Msx1, downregulate Notch signaling pathway target gene Hes1 in cultured eDMCs. Hence, based on the preliminary data and relative studies reported in literature, we hypothesize that DLK1-mediated Notch signaling pathway plays an important role in the regulation of the recovery of odontogenic inductivity in cultured eDMCs through community effect. This study will utilize odontogenic epithelia-mesenchyme recombination experiment etc. to verify the effect of DLK1 treatment and Notch signaling pathway regulation on the odontogenic potential of cultured eDMCs. This study will further elaborate the molecular mechanism of the odontogenic potential regulation of proliferated eDMCs, provide basis to break through the bottleneck, which is to establish method to rescue the odontogenic potential of cultured eDMC through molecular manipulation without contribution from fresh eDMCs.

牙齿缺失是口腔健康的重大问题，利用干细胞通过生物工程技术生成再生牙是重要的研究方向。临床可获取的成牙干细胞数量有限，需体外培养进行扩增。然而扩增细胞成牙潜能消失，这成为研究的最大瓶颈。我们之前的研究表明牙胚间充质的扩增细胞在与新鲜细胞混合产生的群效应下可恢复成牙潜能，其产生机制不明。预实验通过转录组测序分析发现经群效应恢复成牙潜能的扩增细胞的Dlk1表达上调；添加DLK1蛋白可使扩增细胞成牙相关基因Pax9及Msx1表达升高，Notch通路靶基因Hes1表达下降。结合文献报道我们提出假说：DLK1介导Notch通路在群效应恢复扩增细胞成牙潜能的过程中起到调控作用。本研究拟通过上皮-间充质重组成牙实验等方法验证DLK1处理及Notch通路活性调节对扩增细胞成牙潜能的影响，进一步揭示扩增细胞成牙潜能开关的分子调控机制，以期建立无需新鲜细胞参与的分子调控方法恢复扩增细胞成牙潜能，突破研究瓶颈。

牙齿缺失是口腔健康的重大问题，利用干细胞通过生物工程技术生成“再生牙”是重要的研究方向。临床可获取的成牙干细胞数量有限，需体外培养进行扩增。然而扩增细胞成牙潜能消失，这成为研究的最大瓶颈。我们之前的研究表明牙胚间充质的扩增细胞在与新鲜细胞混合产生的群效应下可恢复成牙潜能，其产生机制不明。由相关预实验结果推测，其中参与调控的一个可能的分子机制为新鲜细胞通过分泌DLK1调控扩增细胞内的Notch信号通路激活水平，进而影响扩增细胞中诱导成牙相关基因的表达。为对此进行论证，本项目主要有3个研究内容：1）研究扩增的牙胚间充质细胞，其成牙诱导潜能关闭和重启后，Dlk1的表达、Notch信号通路激活水平以及成牙相关基因的表达水平的变化；2）研究在扩增的牙胚间充质细胞中，DLK1对于Notch信号通路的调控作用，以及DLK1和Notch信号通路对细胞增殖和成牙相关基因的表达的调控作用；3）研究在成牙实验中，调节DLK1和Notch信号通路对扩增的牙胚间充质细胞的成牙诱导潜能的影响。主要研究结果显示，扩增细胞相较于新鲜细胞及恢复成牙诱导潜能的扩增细胞，其信号分子Dlk1表达较低，Notch信号通路活性较高，而成牙相关基因表达较低；另外，添加DLK1以及DAPT对于牙胚间充质细胞能够产生相似的作用效果，即降低细胞增殖速度和Notch通路活性，提高成牙基因表达水平，且作用效果与浓度和时间成正相关。这些结果确定了DLK1对于经体外培养的牙胚间充质细胞的Notch信号通路具有调控作用，并能够以此影响其成牙相关基因的表达。但在诱导成牙的上皮-间充质重组实验中，DLK1/DAPT处理后的扩增细胞与野生型新鲜细胞混合，相较于对照组，既未经药物处理的扩增细胞与野生型新鲜细胞混合，能够诱导牙胚形成的混合细胞中新鲜细胞比例未显著减低；另外，NICD过表达处理后的扩增细胞仍可参与成牙，但相较对照组比例略低。这些结果提示在新鲜细胞使扩增细胞的成牙诱导潜能重启可能是多因素相关，其分子调控机制也可能是众多因子参与的复杂调控系统，需进一步丰富并细化变量进行研究验证。