TGF-β激活的Smad1/5和Smad2/3信号通路正反向调控早期牙胚上皮增殖的研究

The proliferation of dental epithelium is critical for early tooth development. TGF-β/BMP signaling pathways are involved in the early tooth development. It is generally accepted that P-Smad2/3 mediates TGF-β signaling pathway, while P-Smad1/5 mediates BMP signaling pathway in the canonical concept. Our preliminary data showed: P-Smad1/5 was regulated by TGF-β signaling pathway instead of BMP signaling pathway; both Smad2/3 and Smad1/5 control dental epithelial cell proliferation through regulation of Cyclin-D1. Based on these preliminary results, we attend to resolve these following critical scientific questions: ⑴ The molecular mechanisms of Smad1/5 phosphorylation induced by TGF-β during early tooth development; ⑵ Whether TGF-β shows different expression concentration in different areas of dental epithelium which activates different signaling pathways, and makes TGF-β plays positive and negative biological functions; ⑶ At the basalt and central areas, lowly expressed TGF-β is able to activate Smad2/3, then down-regulates Cyclin-D1 expression, which inhibits the proliferation of dental epithelium; ⑷ At those epithelial cells adjacent to dental mesenchyme, the highly expressed TGF-β is able to activate Smad1/5, then up-regulates Cyclin-D1 expression, which promotes the proliferation of dental epithelium. With this study, we can elucidate the regulation mechanism of dental epithelial cell proliferation during early tooth development, and will make the foundation in theory for tooth regeneration and tooth tissue engineering.

牙上皮增殖是早期牙胚发育的关键因素，TGF-β/BMP信号参与早期牙胚发育。经典信号通路认为Smad2/3介导TGF-β信号，Smad1/5介导BMP信号。本课题组前期发现：早期牙上皮内Smad1/5 不受BMP调控，而受TGF-β调控；Smad2/3和Smad1/5通过调控Cyclin-D1影响上皮增殖。基于前期研究基础，本课题拟解决以下关键科学问题：在牙上皮内（1）TGF-β激活Smad1/5的分子机制；（2）TGF-β是否因表达浓度分布不同，激活不同信号通路，发挥不同生理功能；（3）在上皮基底层和中央处是否TGF-β表达较低，激活Smad2/3抑制Cyclin-D1表达，从而抑制细胞增殖；（4）在上皮内邻近间充质的细胞层内是否TGF-β表达较高，激活Smad1/5促进Cyclin-D1表达，从而促进细胞增殖。本研究将进一步阐明早期牙上皮增殖的调控机制，为牙再生和组织工程奠定理论基础。

牙胚发育的启动信号来源于牙上皮，在早期牙胚发育过程中，牙源性上皮增殖是牙胚发育的关键因素。TGF-β/BMP信号在牙发育过程中起诱导和调节作用，被认为是牙胚发育由牙板期进入蕾状期的关键调节分子。经典信号通路认为Smad2/3介导TGF-β信号，Smad1/5介导BMP信号。本课题组前期研究发现在早期牙胚上皮内P-Smad1/5不受BMP调控，而由TGF-β调控。本课题主要研究了在早期牙上皮中TGF-β激活Smad1/5的机制及其生物学功能。本课题通过免疫荧光、原代细胞培养、siRNA干扰、小分子抑制剂、BrdU染色、原位PLA、蛋白免疫沉淀、双荧光素酶报告基因等技术发现：1）在牙胚上皮基底层TGF-β表达浓度较高，可以通过ALK1/2-ALK5复合体激活P-Smad1/5，进而通过促进Cyclin-D1表达促进细胞增殖；2）在上皮中央部位TGF-β表达浓度较低，仅通过ALK5激活P-Smad2/3，进而通过抑制Cyclin-D1表达而抑制细胞增殖；4）TGF-β激活的P-Smad1/5因不能和Smad4结合形成复合体，进而不能激活BMP反应元件，发挥不同于BMP激活的P-Smad1/5的功能，表明在上皮内TGF-β抑制了BMP经典信号通路。本课题研究进一步完善了TGF-β 信号调控牙胚发育的机制，提示TGF-β通过浓度梯度表达不同，在牙胚上皮不同部位激活不同的下游信号分子，从而表现出促进或抑制牙胚上皮细胞增殖，进而参与牙胚上皮的形态发生。本研究进一步阐明了早期牙上皮增殖的调控机制，为牙再生和组织工程奠定理论基础。