炎性微环境下组蛋白去乙酰化酶通过调控miR-17-92家族影响牙周膜干细胞再生能力的机制研究

The regeneration capacity of periodontal ligament stem cells (PDLSCs) was reduced significantly in inflammation microenvironment. It is the main cause of decreased periodontium regeneration. Our previous research demonstrated that the osteogenic differentiation of PDLSCs was inhibited significantly in inflammation microenvironment. It was because that the inflammatory cytokines affected the expression of miR-17. Recently, researchers suggested that the environment could lead to the epigenetic changes. Moreover, the epigenetic could regulate the transcription of microRNA. Interestingly, we also found that the expression pattern of histone deacetylase (HDAC) was changed in inflammatory microenvironment. In addition, the HDAC inhibitors could up-regulate the expression of primary-miR-17 and rescue the osteogenic differentiation of PDLSCs in inflammatory microenvironment. Therefore, we infer that HDAC could affect the regeneration capacity of PDLSCs via modulating the expression of miR-17-92 cluster directly. In this study, we design four part experiments to demonstrated the intrinsic relationship among epigenetic, microRNA and bio-characteristic of stem cells. The experiments include choosing the specific HDAC related with inflammation, the relationship between specific HDAC and bio-characteristics of PDLSCs, the mechanism of specific HDAC regulates the expression of miR-17-92 cluster and the treatment effect of HDAC on periodontitis model in vivo. This study will elucidate the mechanism of the regeneration capacity of PDLSCs decreased in inflammation microenvironment from a new perspective. It will not only explain the regeneration capacity of PDLSCs reduced in inflammation microenvironment, but also provide a new therapeutic target for curing periodontitis.

炎性环境下，牙周膜干细胞（PDLSCs）再生能力下降是导致牙周组织再生异常的重要原因。我们前期证明，炎性环境下PDLSCs中miR-17表达下降是导致其再生能力下降的重要原因。研究表明，表观遗传受环境因素影响且能够调控microRNA的转录。然而表观遗传是否影响炎性环境下PDLSCs中miR-17的表达仍不明确。我们发现，炎性环境中组蛋白去乙酰化酶(HDAC)表达增加，且其抑制剂能够上调pri-miR-17的表达。因此，我们推测HDAC通过调控miR-17-92的转录从而影响PDLSCs的再生能力。本课题拟从炎性环境下变化的特异性HDAC筛选、特异性HDAC与PDLSCs生物学特性的关系、HDAC调控miR-17-92的分子机制，以及动物体内验证四个方面探讨表观遗传、microRNA及干细胞再生能力之间的关系。本课题从新的角度阐明牙周炎再生能力下降的分子机制，并为牙周炎的治疗提供新的思路。

在组蛋白和部分非组蛋白的翻译后修饰中，组蛋白去乙酰化酶（HDACs）家族都起着至关重要的作用。HDAC家族中许多成员在炎症进程中发挥重要作用，然而目前HDAC9在炎症条件下是否被调控和怎样被调控都不清楚。我们的目的在于明确在慢性炎症条件下HDAC9对人牙周膜干细胞成骨分化的影响并探索其调控机制。体外实验中比较健康和牙周炎患者组织中提取的原代牙周膜干细胞(PDLSC)，进行HDAC家族表达检测和HDAC抑制剂以及si-HDAC9干预后的成骨检测。体内实验中使用HDAC抑制剂处理的正常和炎症的牙周膜干细胞膜片移植到裸鼠皮下并检测成骨，或使用HDAC抑制剂治疗LPS诱导的大鼠牙周炎模型并检测骨量等。我们发现炎症抑制牙周膜间充质干细胞的成骨能力并导致HDAC家族中HDAC9的表达水平升高，PDLSC中HDAC9会通过去除miR-17的启动子上乙酰化区域从而抑制miR-17的转录，并抑制了miR-17下游促进成骨细胞成骨的通路，从而导致骨量下降。 最终，HDAC抑制剂可以在体外促进炎症的牙周膜干细胞成骨，在体内促进牙周炎新生骨形成。我们的研究以牙周炎为模型揭示了病程难以逆转的慢性炎症中表观遗传致病机制，为牙周炎治疗提供了新的思路。