Notch信号通路对炎症状态下TGF-β1诱导的牙周膜干细胞向成骨细胞表型转化中作用和机制

Periodontitis is a chronic inflammatory disease affecting the tissues that surround and support the teeth, including gingival inflammation and progressive resorption of the alveolar bone. Most of the therapies which involve the guided tissue regeneration (GTR), could not achieve complete regeneration of periodontal tissues. Periodontal ligament stem cells (PDLSCs) can produce different types of mature cells with a specific phenotype and function.Especially,osteogenic differentiation potential of PDLSCs have brought new light for reconstruction of complex tissue destroyed by periodontitis. In chronic inflammatory bone diseases, such as periodontitis, bone regeneration is inhibited, which may be because the inflammatory microenvironment affects the PDLSCs differentiation. However, the inhibitory molecules repressing bone formation of PDLSCs in inflammatory microenvironment still remain unclear.The main point of this study is to know the role of Notch signaling pathway played in osteogenesis differentiation of PDLCs.By using techniques of conventional molecular biology, quantitative real-time PCR, RNA interference lentiviral infection to understand the effect of Notch signaling axis in periodontal ligament stem cells differentiation, function and migration.The study attempts to answer and resolve the following issues. First, expression characteristics of Notch signaling in inflammatory periodontal tissues and normal periodontal tissues Second, the role of the Notch signaling pathway play in PDLSCs osteogenesis differentiation process under inflammatory microenvironment. Last,the effects and mechanism of Notch signaling pathway in PDLSCs osteogenesis differentiation under inflammatory microenvironment.

牙周炎是以牙龈炎症和牙槽骨吸收为主要特征的慢性炎症性疾病。现有治疗方法引导组织再生术，并不能获得牙周组织的完全再生。牙周膜干细胞（periodontal ligament stem cells, PDLSCs）能产生不同种类的具有特定表型和功能的成熟细胞，特别是其骨向分化的潜能为治疗牙周病造成的牙槽骨缺损提供了新思路。然而炎症状态下PDLSCs骨向分化过程的分子机制及调控方式尚不清楚。本研究从Notch信号通道对炎症状态下TGF-β1诱导牙周膜干细胞向成骨细胞表型转化的功能调控入手，采用分子生物学技术、实时定量PCR、慢病毒转染的RNA干扰技术等技术，试图回答和解决：①炎性牙周组织和正常牙周组织中Notch信号的表达特性；②炎性环境下Notch信号在PDLSCs向成骨细胞分化过程中的表达特性；③调控Notch信号通路对炎症环境下TGF-β1诱导PDLSCs向成骨细胞分化功能的影响及机制。

牙周炎是以牙龈炎症和牙槽骨吸收为主要特征的慢性炎症性疾病。牙周膜干细胞（periodontal ligament stecells, PDLSCs）骨向分化的潜能为治疗牙周病造成的牙槽骨缺损提供了新思路。本课题以牙周膜干细胞成骨分化过程中，Notch信号通路、TNF-α及TGF-β1对其功能调控入手，运用多种研究方法包括干细胞鉴定、实时定量PCR技术，Weston Blot实验、免疫组化、慢病毒转染的RNA干扰技术等，开展研究工作得出以下结果。1）对正常组织和炎性组织来源的PDLSCs形态学和细胞表面标记物检测显示两者间无明显差异，提示PDLSCs的存在和形态不受炎性环境的影响。炎症来源的牙周膜干细胞可能抑制了Notch 信号分子的表达，低水平的Notch 信号的激活可能有利于牙周炎缺损牙槽骨的成骨分化。2）TNF-α能够促进PDLSCs增殖而抑制其骨向分化过程中成骨相关基因的表达，同时抑制Notch信号通路相关分子的表达，说明TNF-α能够抑制Notch信号通路的表达，通过其调控PDLSCs骨向分化。3）通过慢病毒转染NICD的PDLSCs其增值及迁移能力加强，均快于空载病毒的PDLSCs。4）TNF-α对过表达的NICD/PDLSCs成骨分化能力有抑制作用。5）TGF-β1能促进PDLSCs的增殖，并提高PDLSCs的成骨分化能力，Notch信号通路中的Notch1、Notch2、JAG1、JGA2、Hes-1的表达水平明显降低，且其作用与TGF-β1的浓度有关。6）DAPT可抑制Notch信号通路的传递，从而抑制PDLSC增殖及骨向分化，同时DAPT浓度为50 μmol/L时抑制能力最强。本项目是针对炎性因子、Notch信号通路相关机制展开的研究，为牙周炎引起的牙槽骨缺损修复的调控提供重要的理论价值。