内质网应激在牙周炎性环境影响牙周膜干细胞功能中的机制研究

Alveolar bone destruction caused by periodontitis is hard to be restored, which is the main reason for teeth missing. The key point of prevention lies in the transformation of the course of inflammatory destruction into tissue regeneration process. Periodontal ligament stem cells (PDLSCs) is the main cell resource of maintaining periodontal tissue structure as well as regeneration. Our previous study indicated a huge difference between PDLSCs of different status. Specifically, the osteogenic differentiation is significantly weakened in inflammatory microenvironment , and the expression of osteoblast-related genes decreased too. It is estimated that PDLSCs lost part of the stem cell characteristics and the regeneration properties under inflammatory microenvironment. However, its influencing mechanisms and pathways is not yet clear. Endoplasmic reticulum stress (ERs) is a protective response in eukaryotic cells which has been found in recent years and influence many intracellular signaling molecules, it is also proved to be closely associated with the initiation and development of inflammation. But the possible connection between ERs and PDLSCs has not been reported. Our previous in vitro studies found that the expression of endoplasmic reticulum stress marker protein GRP78, CHOP were significantly increased in PDLSCs. This project intends to further and comprehensively study the regulatory ways and mechanisms by which endoplasmic reticulum stress affect the osteogenic properties of PDLSCs, then figure out the key targets. The next work will aim to restrain the destruction of periodontitis through ERs regulation, and to provide a new theoretical and experimental basis to the promotion of tissue restoration and reconstruction.

牙周炎造成的牙槽骨破坏重且难以自行修复，是缺牙的主要原因。如何逆转病程为组织再生是防治关键。牙周膜干细胞（PDLSCs）是维持牙周组织结构和再生修复的主要功能细胞来源。我们前期研究发现不同状态的PDLSCs存在巨大差异，表现为炎症时其成骨分化能力明显减弱，成骨相关基因表达下降；说明炎性微环境下PDLSCs部分失去了干细胞特性和牙周组织再生能力。但其影响机制和作用途径并不清楚。内质网应激(ERs)是近年发现的一种真核细胞保护性反应；对众多胞内信号分子发挥影响，与炎症的发生发展密切相关。但ERs对PDLSCs的影响方式、程度等均无报道。我们前期研究发现炎性刺激下PDLSCs内质网应激标志蛋白GRP78、CHOP表达水平显著升高。本课题拟进一步全面研究内质网应激影响PDLSCs成骨分化的调控途径和机制，找出关键靶点，为通过ERs调控来抑制牙周炎的破坏，促进组织修复重建提供新的理论依据和实验基础。

牙周炎造成的牙槽骨破坏重且难以自行修复，是缺牙的主要原因。如何利用牙周膜干细胞（PDLSCs）逆转病程并使组织再生是新的发展方向之一。炎性微环境下PDLSCs部分失去了干细胞特性和牙周组织再生能力，但其影响机制和作用途径并不清楚。内质网应激(ERs)与炎症的发生发展密切相关，但ERs对PDLSCs的影响方式、程度等均无报道。本课题即研究ERs影响PDLSCs成骨分化的调控途径和机制，找出关键靶点，尝试为抑制牙周炎的破坏，促进组织修复重建提供新的理论依据和实验基础。我们从健康、牙周炎等不同来源组织中分离培养PDLSCs，并进行鉴定；或通过模拟炎性环境刺激PDLSCs，检测结果均证实炎症环境下PDLSC的成骨能力有明显降低；健康人及不同类型牙周病变患者牙周组织中ERs表达有差异；不同方式炎症刺激下也均可以诱导PDLSC发生ERs；并进而可抑制PDLSC 的成骨分化。炎症微环境下ERs的UPR通路相关因子表达发生变化；干预阻断ERs后，PERK通路相关因子的基因和蛋白表达发生变化，并使PDLSC的成骨能力得到回升。 与ERs相关联的组蛋白乙酰转移酶（HATs）在健康和炎症不同来源的PDLSCs中表达有差异；分别抑制或过表达升高不同HATs后，UPR信号分子的表达也发生变化；其中，无论模拟短期、长期炎症微环境，MORF表达均有变化；而对MORF干预后，PDLSCs的成骨分化功能发生逆转。对小动物的体内干预试验进一步验证，使用PERK通路抑制剂可以部分阻断大鼠实验性牙周炎的牙槽骨缺损。概括而言，本研究的科学发现意义在于：证实牙周炎状态下，牙周组织(牙龈、牙周膜)的UPR水平升高；炎症微环境下，PDLSCs内质网应激水平升高；无论用UPR抑制剂或者通过干扰RNA技术均可恢复炎症环境下PDLSC被降低的成骨分化能力；组蛋白乙酰转移酶MORF可以通过转录抑制作用影响UPR重要分子PERK启动子的转录活性；而炎症微环境可以通过降低MORF引起UPR激活，进而影响PDLSCs的成骨功能。