Ezh2调控牙髓损伤修复的分子机制

Dental pulp stem cell differentiation is essential to dental pulp repair. Epigenetic regulation plays an important role in stem cell fate decision. Ezh2, a histone methylthansferase, is crucial to "stemness" of stem cells. Ezh2 was found to be down-regulated during mineralization of mesenchymal stem cells, by modulating the nuclear transcriptional factor. Our preliminary data showed that Ezh2 expression in dental pulp cells were decreased by LPS treatment and hypoxia, which suggesting its role in dental pulp injury and repair. This project was to study the biological effect of Ezh2 on dental pulp stem cell differentiation by overexpressing or silencing Ezh2 in cells, its enzymatic activity involvement during the process by construct its mutation form without enzymatic activity, its target genes by transignal protein/DNA combo array, its regulation on the target gene by fluorescence reporter and CHIP assay. In vivo mineralization experiment was used to test the effect of Ezh2 on dental pulp stem cells. This project will help to elucidate the role of epigenetic control in dental pulp injury and repair, as well as the theory of dental pulp biology.

调控牙髓干细胞的分化是牙髓损伤修复的关键，表观遗传调控在干细胞命运决定中具有十分重要的作用。Ezh2是一种组蛋白甲基转移酶，与干细胞的分化密切相关。Ezh2在间充质干细胞矿化过程中表达下调，通过调控核转录因子的表达调控其分化。我们前期研究发现，炎性刺激下牙髓细胞中Ezh2的表达下调，提示其参与了牙髓损伤修复中的分子细胞事件。本项目在此基础上，通过构建Ezh2过表达和沉默质粒，观察其对牙髓干细胞增殖分化的影响；同时通过构建Ezh2酶活性缺失的质粒，研究Ezh2的组蛋白甲基转移酶活性是否为这个过程所需；采用转录因子活性芯片，结合生物信息学分析，筛选Ezh2在牙髓干细胞中调控的靶基因，并通过荧光素酶报告基因和染色质免疫共沉淀技术研究调控机理；通过基因修饰牙髓干细胞体内矿化实验验证Ezh2在牙髓损伤修复中的作用。本项目的实施有助于阐明牙髓损伤修复过程中表观遗传调控因子的作用，充实牙髓生物学理论。

调控牙髓干细胞的分化是牙髓损伤修复的关键，表观遗传调控在干细胞命运决定中具有十分重要的作用。Ezh2是一种组蛋白甲基转移酶，Ezh2不仅参与调控不同细胞的增殖和分化，还参与调控牙髓炎症修复过程。本项目构建了Ezh2过表达、Ezh2酶活性缺失和Ezh2沉默病毒，观察其对牙髓干细胞炎症、增殖、分化的影响，及影响机制。结果发现牙髓炎症时Ezh2表达下调，Ezh2可以通过调节炎性因子表达而促进牙髓炎症发生，Ezh2抑制剂则可以减轻牙髓炎症程度；Ezh2沉默后，牙髓干细胞的增殖能力明显下调，机制研究发现Ezh2不通过组蛋白甲基化转移酶活性功能调控牙髓干细胞进入细胞增殖周期，而通过调控PCNA和DNA聚合酶δ的结合来调控DNA扩增过程；Ezh2沉默后，牙髓干细胞的骨向分化能力明显增强，机制研究发现Ezh2通过甲基化转移酶活性调控β-catenin的的表达，调控WNT经典信号通路，从而调控分化过程。本项目首次提出Ezh2抑制剂可能成为临床上用来抑制牙髓炎症的药物，为牙髓炎治疗开辟了研究方向；首次发现Ezh2可以通过调节PCNA和DNA聚合酶δ的结合，从而调节DNA合成；Ezh2可以通过调节β-catenin的转录，调控牙髓干细胞的分化过程，有助于阐明牙髓损伤修复过程中表观遗传调控因子的作用，充实牙髓生物学理论。