小型猪牙齿发育早期形态发生关键microRNA的发现及其功能分析

The whole tooth regeneration based on the development model is a very important issue in oral medical research field, which must be based on the further understanding of the mechanism of tooth development and tooth morphogenesis. Recent studies had shown that microRNA (miRNA) may be play important roles in the regulation of tooth development and tooth morphogenesis. In the previous studies, we chose large mammals "miniature pig" as an animal model, conducted a large-scale screening for miRNA genes of miniature pig's four kinds of teeth in 3 main time points by using miRNAs expression profiles in the early tooth development, and predicted 5 miRNA as the valuable candidate list of tooth morphogenesis related miRNA. Base on this data, this topic using lentivirus infection method to make the candidate miRNA excessive expression or been inhibited in the tooth germ, so that to determine "key miRNA" in the tooth morphogenesis of miniature pig teeth. And then through the real time PCR chip, Western blot and fluorescence immune chemistry methods to elucidate its mechanism in the teeth development process .Finally using the tooth regeneration model based on tooth germ reorganization and solution, screen out the miRNAs which could promote the teeth regeneration from "key miRNA". In order to rich miRNA research material in the field of teeth development and regeneration, and establish theoretical foundation of miRNA promote tooth regeneration. And in the future of biological engineering tooth regeneration process, find a useful miRNA regulation tooth morphogenesis technology strategy.

基于发育模式的全牙再生是牙再生策略之一，其基础是对牙发育机理，包括对牙齿发育早期形态发生调控机制的深刻认知。有研究证实，miRNA在牙齿发育尤其是形态发生中发挥着重要的作用。我们前期用"小型猪"作为模型动物，对4类牙齿在3牙胚早期发育主要时间点的miRNA，进行了表达谱芯片分析，初步确定了5个可能与牙齿形态发生相关的候选miRNA。本课题拟在此基础上，用慢病毒转染系统使候选miRNA在牙胚中过表达或被抑制，以确定其是否为牙齿形态发生的关键miRNA；进而通过qRT-PCR芯片、Western blot及荧光免疫化学等方法阐明其作用机制；最后通过基于牙胚解离重组的牙再生模型，从中筛选出可促进牙齿再生的miRNA。希望丰富miRNA在牙齿发育和再生领域的研究资料，为基于发育模式的牙齿再生提供一些理论依据；并在未来的生物工程牙再生过程中，探索出一种利用miRNA调控牙齿形态发生的技术策略。

基于发育模式的全牙再生是牙再生策略之一，其基础是对牙发育机理,包括对牙齿发育早期形态发生调控机制的深刻认知。有研究证实,miRNA在牙齿发育尤其是形态发生中发挥着重要的作用。我们前期用“小型猪”作为模型动物,对 4 类牙齿在 3 牙胚早期发育主要时间点的miRNA,进行了表达谱芯片分析,初步确定了 5 个可能与牙齿形态发生相关的候选 miRNA。本课题在此基础上,首先利用Real-Time PCR 验证了基因芯片的结果，证实五个候选miRNA在牙齿发育形态发生时期的表达情况，并且进一步确定其表达的组织特异性。然后利用慢病毒转染系统构建候选miRNA 的过表达及表达抑制载体, 结合牙胚组织三维培养系统，将候选miRNA分别在小型猪牙齿形态发生时期的4类牙齿中过表达及表达抑制，以确定其是否为牙齿形态发生的关键 miRNA；功能验证表明，ssc-mir-133b在E40前磨牙过表达后，能够抑制牙胚正常发育；体内实验证实，过表达ssc-mir-133b的前磨牙牙胚在裸鼠的皮下不能正常发育。体外机制研究表明，ssc-mir-133b通过下游Bcl-2家族抗凋亡蛋白Mcl-1发挥作用，过表达的ssc-mir-133b通过抑制Mcl-1的抗凋亡作用，启动下游内源性凋亡机制，牙胚最终发育失败。本研究通过对候选miRNA在小型猪牙齿发育形态发生阶段进行功能学及机制方向的探讨，丰富了 miRNA 在牙齿发育和再生领域的研究资料，为基于 发育模式的牙齿再生提供了一些理论依据；希望基于本研究的结果，能够帮助我们在未来的生物工程牙再生过程中，探索出一种利用 miRNA 调控牙齿形态发生的技术策略。