褪黑素及其受体的节律活动调节小鼠牙胚发育的机制研究

Abnormal tooth development is very common in clinic. The treatment is complicated and time consuming. Since the etiology of the abnormality is not clear, it is difficult to develop any preventive strategy for the disease. Recently, it has been shown that tooth growth is remarkably rhythmic. While melatonin (MT) plays a pivotal role in day/night rhythms in mammals, its function on tooth development has never been investigated. Our preliminary study has shown that MT receptors (MRs) are highly expressed in mouse tooth germ. More interestingly, we have discovered that changing light/dark cycle significantly affected expression levels of MRs in the tooth germ. Hence, the applicant hypothesizes that activity of MT and MR system may play a role in tooth development. Malfunctioning of this system may attribute to abnormal tooth development. To test this hypothesis, the present study will investigate the correlation between MT/MR rhythmic activity and the rhythmic growth of teeth during development at molecular and ultrastructural levels. Furthermore, we will disturb the rhythmic activity of MT/MR system in pregnant mice by altering the day/night cycle or treating the mice with MR antagonist, which will demonstrate the effect of rhythmic activity of MT/MR system on tooth development. By comparing changes in signal pathways of tooth germ between animals with disturbed rhythm and normal rhythm, we may reveal the cellular and molecular mechanisms responsible for this regulation, which may throw lights on the etiology of abnormal tooth development and suggest potential strategies for prevention.

牙齿发育异常临床上极为常见，治疗过程漫长复杂，且因发病机制不清，很难预防。近年研究证实，牙齿发育具有明显的节律性。褪黑素（melatonin，MT）是哺乳动物调节机体昼夜节律性生长发育的关键激素，但它是否直接参与牙齿发育的调控仍不清楚。课题组预实验发现：MT受体在小鼠牙胚中呈阳性表达，而且改变孕鼠的昼夜节律可显著影响新生小鼠牙胚MT受体水平。据此课题组假设：MT及其受体的节律改变可能是牙齿发育异常的机制之一。为充分证实该假设，我们在分子水平（基因和蛋白）及超微结构水平验证正常小鼠中MT及其受体的节律性变化与牙齿节律性发育之间的关联、MT调控模型（改变孕鼠昼夜节律或给予MT受体拮抗剂）小鼠中MT及其受体的节律改变对小鼠牙齿发育的影响，以及对比MT调控模型与正常小鼠牙胚的发育，筛查MT参与调控牙齿发育的关键信号通路及分子机制，为阐明牙齿发育异常的发病机制及防治提供新的理论依据。

牙釉质中体现昼夜节律性的生长线以及褪黑素受体的存在已有文献报道，而我们假设牙釉质的昼夜节律性生长是受褪黑素受体调控的。为了验证该假设，我们在新生小鼠的下颌第一磨牙牙胚中检测褪黑素受体以及AMELX的表达。通过qRT-PCR和免疫组化的方法，我们发现小鼠的下颌第一磨牙牙胚中褪黑素受体和AMELX的表达在出生后明显增加，在出生后第3和第4天达到高峰，之后减少。在全天24小时黑暗饲养以及全天24小时光照饲养的新生小鼠中，免疫组化检测下颌第一磨牙牙胚中褪黑素受体和AMELX的表达，发现表达有明显的延迟。而且，牙釉质发育在组织学形态上也表现出明显的不成熟，尤其是那些出生后即被24小时全天光照处理的小鼠。孕鼠的昼夜节律被干扰也会导致其分娩产下的新生小鼠的牙胚发育迟缓。对孕鼠进行4P-PDOT（一种褪黑素受体阻断剂）的注射会导致新生小鼠牙胚中褪黑素受体以及AMELX表达降低以及牙釉质发育的缺陷。电镜下显示的核固缩明显增多以及釉质基质发育的缺陷均能证明。因此，出生前以及出生后的正常昼夜节律对牙釉质的正常发育都是非常重要的。