GREM1-BMP信号通路对根尖牙乳头干细胞定向分化及牙齿组织再生功能的影响及调控机制研究

Root hypoplasia and regeneration is a key problem in oral treatment for children, however, the traditional therapy couldn’t reach the satisfied therapeutic effect. Stem cell mediated root regeneration bring hope but still have many problems, such as limited seed cells, poor efficacy, etc. Stem cells niche play the key role for homeostasis and tissue regeneration. Our previous studies found that BMP2, BMP5, BMP6, BMP7 are down-regulated in stem cells from apical papilla (SCAPs) than those in the tissue of apical papilla, but the antagonist of bone morphogenetic proteins-GREM1 is up-regulated. And in vitro experiments showed that BMP6 could significantly enhance the proliferation and differentiation potentials of SCAPs, and BMP2, BMP6, and BMP7 negatively regulated by GREM1, suggesting that GREM1-BMP signaling pathway may play an important role to regulate the stem cells function and tooth regeneration. Previous study found that GREM1 could bind with YWHAH, YWHAH could associated with YAP, and YAP could regulate the BMP transcript and the differentiation function of stem cells, indicated that GREM1/YWHAH/YAP might form protein complex, and regulate BMP2, BMP6, and BMP7 and stem cells function. But their function and molecular mechanism is not clear. In this project, we want to investigate the biological regulation function of GREM1/YWHAH/YAP-BMP2/6/7 molecules in SCAPs by huamn recombinant protein, virus mediated gene knockout and over-expression. And then by the loss of function and gain of function studies, the effect of GREM1/YWHAH/YAP-BMP2/6/7 molecules in SCAPs mediated dental root regeneration in nude mice will be elucidated. Finally, we will figure out the molecular mechanism to explain how GREM1/YWHAH/YAP regulate BMP2/6/7 in SCAPs and dental root regeneration. Taken together, this study will illuminate the mechanism of the special stem cells niche to regulate the stem cells function, provide the key target genes and certain theoretical basis to reconstruct the genes expressions in stem cells niche, in order to promote stem cells mediated dental tissue regeneration.

干细胞介导的牙根再生是牙根发育不完全治疗的未来发展防线，但存在再生效果不佳等问题。微环境是维持干细胞功能的重要因素，课题组前期发现根尖牙乳头干细胞微环境中BMP2、5、6和7高表达，而其拮抗基因GREM1低表达；GREM1可以定位于细胞核内，负调控BMP2、6和7；以往研究还发现GREM1与YWHAH结合、YWHAH与YAP结合；而YAP具有调控BMP转录及干细胞分化的功能。提示GREM1/YWHAH/YAP可能具有调控多个BMP信号分子转录及干细胞功能的重要作用。本课题拟利用重组蛋白、病毒转染等技术研究揭示GREM1及BMP2、6和7对根尖牙乳头干细胞定向分化功能及牙根再生的影响，阐明GREM1/YWHAH/YAP对BMP信号分子的调控机制。该研究将有助于揭示微环境对干细胞的功能影响及分子机制，为模拟并重建微环境中基因表达，促进干细胞功能及其介导的牙根再生提供理论依据和关键靶点。

干细胞介导的牙根再生是牙根发育不完全治疗的未来发展防线,但存在再生效果不佳等问题。微环境是维持干细胞功能的重要因素,课题组前期发现根尖牙乳头干细胞微环境中BMP2、5、6和7高表达,而其拮抗基因GREM1低表达;GREM1可以定位于细胞核内,负调控BMP2、6和7;以往研究还发现GREM1与YWHAH结合、YWHAH与YAP结合;而YAP具有调控BMP 转录及干细胞分化的功能。提示GREM1/YWHAH/YAP可能具有调控多个BMP信号分子转录及干细胞功能的重要作用。本课题拟利用免疫共沉淀技术、生物信息学分析、获得性及丧失性功能研究及动物体内研究揭示GREM1对干细胞定向分化功能及生物牙根再生的影响，阐明GREM1对BMP信号分子的调控机制。体外功能实验结果显示在脂肪干细胞中,过表达 GREM1抑制其成骨分化及衰老，敲除 GREM1促进脂肪干细胞的成骨分化功能及衰老。机制研究方面，IP实验结果显示， GREM1与YWHAH、YAP可以形成蛋白复合体，蛋白之间存在着作用。并且GREM1可以拮抗 BMP2、BMP6 和 BMP7 信号分子的转录，YWHAH可以协同 BMP2、BMP6 和 BMP7 信号分子的转录。该研究将有助于揭示微环境对干细胞的功能影响及分子机制,为模拟 并重建微环境中基因表达,促进干细胞功能及其介导的牙根再生提供理论依据和关键靶点 。