ASH1L调控牙髓干细胞命运选择的机制研究

Dental pulp regeneration depends on the differentiation of dental pulp stem cells into odontoblast-like cells，forming reparative dentin to protect pulp from the stimulus. Differentiation of dental pulp stem cell toward odontoblast-like cells is the key step in pulp repair. Epigenetics has been found to be more and more important for stem cell fate decision. ASH1L is a histone methyltransferase, which was shown to activate gene expression through antagonizing PcG-mediated abnormal silence. We previously found ASH1L was obviously increased using expression profiling of epigenetic regulators in hypoxia-induced human dental pulp cells. ASH1L expression was also elevated during osteogenic differentiation of HDSCs. ASH1L silence eliminated dental pulp mineralization ability, by suppressing osteogenic gene and nuclear transcription factor expression such as RUNX2. Based on these in vitro results, we hypothesized that ASH1L might play pivotal role in pulp damage and repair. This project was aimed to study the in vivo effect of ASH1L on the mineralization of HDSCs by overexpression method. By constructing its mutation form without enzymatic activity, we plan to test the necessity of its enzymatic activity in regulating HDSCs differentiation. Besides, we plan to using CHIP and other techniques to test the underlying mechanism of ASH1L in regulating the osteogenic/odontogenic differentiation related genes expression. This project will highlight a mechanism by which epigenetic modifications control stem cell fate, enriching the biological theories of dental pulp and shed new light for dental pulp regeneration.

牙髓干细胞的成牙向分化是牙髓损伤修复的关键步骤，表观遗传修饰是近年来干细胞命运调控研究的新热点。ASH1L是重要的组蛋白甲基转移酶，被认为通过拮抗PcG蛋白家族激活基因的表达。课题组前期通过基因芯片发现，牙髓干细胞缺氧和体外矿化诱导时ASH1L表达明显升高，敲低ASH1L则牙髓干细胞的体外矿化能力降低，提示其在牙髓前体细胞成牙向分化过程中有重要作用。本课题拟在此基础上，通过基因修饰牙髓干细胞体内矿化实验验证ASH1L在牙髓干细胞成牙向分化中的作用；通过构建ASH1L过表达及酶活性缺失质粒，研究ASH1L是否通过其酶活性发挥调控作用；通过染色质免疫共沉淀等技术研究其是否结合于RUNX2等核转录因子从而调控其转录，进而发挥其调控牙髓干细胞命运选择的作用。本课题的实施有助于阐明表观遗传在间充质干细胞命运选择中的作用，并充实牙髓生物学理论，为牙髓损伤修复治疗提供新的选择和思路。

牙髓干细胞的成牙向分化是牙髓损伤修复的关键步骤，表观遗传修饰是近年来干细胞命运调控研究的新热点。本项目主要研究了H3K4三甲基转移酶ASH1L在牙髓炎症及间充质干细胞命运选择中的生物学功能及作用机制。从ASH1L在牙髓炎症及牙髓细胞分化过程中的表达模式入手，通过干预ASH1L（过表达、敲低、酶活性突变），观察其对牙髓细胞炎症、分化的影响及机制。结果发现，ASH1L 在人牙髓炎组织及大鼠实验性牙髓炎模型中表达上调；在TNFα刺激的牙髓细胞中，ASH1L 基因沉默可促进基质金属蛋白酶（MMPs ）的表达及酶活性；P38、ERK通路的抑制影响了ASH1L沉默对MMP1、MMP2、MMP13蛋白表达及酶活性的诱导作用，表明在牙髓炎中ASH1L可能通过MAPK信号通路调控MMPs；ASH1L促进了间充质干细胞细胞（DPSCs 和 C3H10T1/2 ）的成骨分化、成软骨分化，抑制了间充质干细胞的成脂肪分化，而ASH1L酶活性突变的ASH1LΔN对成骨、成软骨及成脂肪能力无明显影响，表明ASH1L对MSCs分化的调控作用依赖于其H3K4甲基转移酶活性；ASH1L可直接与Hoxa10、Osx、Runx2、Sox9基因启动子区结合，上调H3K4me3修饰水平，从而促进上述基因转录，调控成骨、成软骨分化；ASH1L调控Pparγ抑制基因Creb的H3K4me3水平，从而抑制了成脂肪分化。本项目首次阐明ASH1L在牙髓炎中的作用，并首次对ASH1L在间充质干细胞分化过程中的调控作用及相关分子机制进行了初步研究。本课题的实施有助于阐明表观遗传在间充质干细胞命运选择中的作用，并充实牙髓生物学理论，为牙髓损伤修复治疗提供新的选择和思路。