SFRP2甲基化调控在低氧微环境中对牙源性间充质干细胞功能的影响及调控机制研究

The biological regeneration and restoration of tooth defect and loss is the key scientific issue in dental field. By now, the mesenchymal stem cells (MSCs) mediated dental tissues regeneration is the hot topic in dental research field, but the molecular mechanism about niche, such as hypoxia, which regulated the MSCs function in Jaw is unclear; this restrict the potential MSC study and application. SFRP2, is a soluble Wnt inhibitor, directly binds to Wnt and block the activation of Wnt signaling. Previous report discovered that SFRP2 increased the survival of MSCs under hypoxia. In our previous study, we found that SFRP2 transcription was regulated by histone demethylase, and SFRP2 could enhance the osteo/dentinogenic differentiation potential in MSCs. Then hypoxia inhibited the expression of SFRP2, decreased expression of SFRP2 induced the apoptosis of MSCs. These results suggested that SFRP2 might play the important role for regulating the directed differentiation and regeneration potential of MSCs under hypoxia, but the function and molecular mechanism is not very clear. In this project, we want to investigate the biological regulation function of SFRP2 for directed differentiation of MSCs by lentivirus mediated gene knockout and over-expression. And then by mimic the clinic conditions, the effect of SFRP2 in MSC mediated dental tissues regeneration in miniature pigs will be elucidated. Finally, we will elucidate the molecular mechanism to explain how methylation of SFRP2 regulate the function of MSCs and dental tissue regeneration. In conclusion, our investigation will discover the effect of jaw microenvironment on the function of MSCs and its molecular mechanism, and provide insight into the mechanisms responsible for improving MSC-mediated dental tissue regeneration approaches in clinic.

干细胞介导的牙齿组织再生是目前口腔医学研究的热点，但在低氧颌骨特定微环境下干细胞功能调控的分子机制尚不清楚，从而影响了其应用。SFRP2是一个可溶性的WNT信号调节因子，具有低氧下抗细胞凋亡作用。本课题组前期研究发现SFRP2受组蛋白去甲基化酶调控，可以促进间充质干细胞成骨/成牙分化功能，低氧降低SFRP2表达，SFRP2低表达易致干细胞凋亡。但SFRP2在低氧环境下对牙源性间充质干细胞（MSCs）分化与再生功能的影响与机制尚不清楚。本课题拟通过丧失性及获得性功能研究揭示SFRP2在低氧下对牙源性MSCs定向分化的影响，利用小型猪动物模型研究SFRP2对牙源性MSCs介导的牙齿组织再生效果, 利用免疫共沉淀技术、CHIP方法揭示低氧下SFRP2甲基化调控调节干细胞功能的分子机制。研究结果有助于阐明颌骨特定微环境对MSCs功能的影响及分子机制，为临床促进干细胞介导的牙齿组织再生提供实验依据。

SFRP2是一个可溶性的WNT信号调节因子，具有低氧下抗细胞凋亡作用。本课题组前期研究发现SFRP2受组蛋白去甲基化酶调控，可以促进间充质干细胞成骨/成牙分化功能，低氧降低SFRP2表达，易致干细胞凋亡。但SFRP2在低氧环境下对牙源性间充质干细胞（MSCs）分化与再生功能的影响与机制尚不清楚。本课题拟通过丧失性及获得性功能研究揭示SFRP2在低氧下对牙源性MSCs定向分化的影响；研究表明SFRP2促进根尖牙乳头干细胞（SCAPs）在低氧和炎症环境下成骨／成牙向分化能力和旁分泌能力。利用免疫共沉淀技术、CHIP方法揭示低氧下SFRP2甲基化调控调节干细胞功能的分子机制；（1）SFRP2通过拮抗经典Wnt信号通路促进SCAPs的成骨向分化：SFRP2通过促进磷酸化和降低核β-catenin在体内外的表达来抑制典型Wnt信号传导。SFRP2下调Wnt信号通路的靶基因AXIN2（axin相关蛋白2（protein 2）和基质金属蛋白酶7（MMP7）的表达。SFRP2能挽救SCAPs由WNT1受损的的成骨分化潜能；（2）SFRP2通过抑制经典Wnt/β-catenin信号通路，从而抑制靶基因的NF-κB信号通路。炎症或缺氧条件可促进赖氨酸脱甲基酶2A（KDM2A）的表达，通过降低SFRP2组蛋白甲基化来抑制SFRP2的转录。通过小型猪建立动物牙周炎模型，研究SFRP2对牙源性MSCs介导的牙周组织再生效果；建立小型猪牙周炎模型，随机分为3组，分别在缺损部位注射过表达SFRP2的SCAPs、Vector转染的SCAPs和无菌生理盐水。分别在实验前、建模后（-4W）、治疗后3个月（12W）进行牙周临床指标（PD、AL、GR）和CT影像学检查，组织学观察牙周组织再生情况，结果表明：SCAPs和经SFRP2感染的SCAPs，治疗后均能改善小型猪牙周炎状况，效果明显优于对照组，其中经SFRP2过表达的SCAPs治疗组效果最好。通过该项目发现SFRP2体外促进根SCAPs在低氧和炎症环境下成骨／成牙向分化能力和旁分泌能力，体内促进SCAPs介导的小型猪牙周组织再生，并发现SFRP2通过拮抗经典Wnt信号通路促进SCAPs的成骨向分化。研究结果有助于阐明颌骨特定微环境对MSCs功能的影响及分子机制，为临床促进干细胞介导的牙齿组织再生提供实验依据。.