牙周来源的间充质细胞膜片促进炎症状态下牙周组织再生及相关机制研究

Periodontitis is a serious disease which influence human oral and systemic health. It was confirmed that the regenerative potential of periodontal ligament cells was effected by low inflammatory microenvironment of periodontal tissue. In cell sheet, cells are protected by rich extracellular matrix and the important information transmission and exchange moleculars were also preserved to enable that cells could make a integral defense to the external stimulus. Therefore, the periodontal regeneration potential of cell sheets in low inflammatory microenvironment should be more advantageous than simple cells. But it is unkonwn about the capability of cell sheets on inflammatory defense and immunity regulation. Our previous study found that dental follicle cell sheet was more effective and suitable for periodontal tissue regeneration than periodontal ligament sheet. In addition, dental follicle cells are derived from the mesenchymal stem cells of the developmental dental germ, and the minimal immunogenicity is a great advantage. This project is to set up LPS-induced inflammatory microenvironment in vitro and construct periodontitis in beagle dogs in situ, to investigate the influene of low inflammatory microenvironment on the biological characteristics of dental follicle cell sheets, the effect of dental follicle cell sheets promoting periodontal regeneration and the mechanisms of inflammatory defense and immunity regulation. It could provide the theoretical foundation for periodontal repair and regeneration, promote the progress of the clinical treatment of periodontitis, and explore the significance of stem cell therapy treating for human diseases.

牙周炎是严重影响人类口腔及全身健康的重要疾病，研究证实牙周的低炎症微环境抑制牙周膜细胞的再生潜能。细胞膜片依靠丰富的细胞外基质将细胞包裹起来，并保留了细胞之间的信息传递和交流，对外界刺激可以做出整体防御，在低炎症微环境中细胞膜片的牙周组织再生潜能应该优于单一的细胞，但是目前对细胞膜片抵御炎症和调控免疫反应方面的情况尚不清楚。本课题组前期研究证实牙囊细胞膜片比牙周膜细胞膜片具有更强的牙周组织再生能力，而且牙囊细胞是来源于牙胚发育期的间充质干细胞，较低的免疫原性使其具有突出优势。本课题拟体外模拟炎症微环境和构建比格犬牙周炎模型，探讨炎症微环境对牙囊细胞膜片生物学特性的影响，以及炎症状态下牙囊细胞膜片促进牙周组织再生的效果及其对机体免疫炎症反应的影响和调控机制，为牙周组织的修复重建和再生提供理论依据，促进牙周炎临床治疗技术的进一步发展，探索干细胞治疗在人体疾病治疗方面的重要意义。

牙周炎的临床治疗是在一个低炎症刺激的微环境中进行的。牙周基础治疗后血清中炎症因子的水平仍高于正常人群，异常表达的炎症介质在修复再生过程中对组织的毒性作用持续存在。因此，了解牙周炎症微环境对干细胞修复再生能力的影响和作用机制对获得完全的牙周组织再生具有十分重要的意义。.本课题旨在通过牙龈卟啉单胞菌的脂多糖（Lipopolysaccharide from Porphyromonas gingivalis，P.g. LPS）体外模拟炎症微环境，并且构建比格犬牙周炎缺损模型，探索炎症微环境对DFC膜片和PDLC膜片生物学特性的影响和可能机制，以及炎症微环境下细胞膜片促进牙周组织再生的效果。.实验证实：1. LPS对两种细胞膜片的分化产生了不同的作用：LPS的刺激使PDLC膜片所有基因表达均下降，特别是明显削弱了的成骨向分化优势。PDLC膜片原本具有优势的矿化相关基因BSP、RUNX2、OCN和OPN在LPS刺激后表达大幅下调；LPS刺激DFC膜片后虽有矿化相关基因（ALP和OCN）、成牙骨质相关基因（CP23）和COL 表达下调，但也有部分矿化相关基因BSP、OPN表达上调，牙周膜相关基因PERIOSTIN表达上调，对粘附相关基因的表达无影响。总的来说，除了BSP、在DFC膜片+LPS组低于PDLC膜片+LPS组或CP23在两组间无差异外，其余基因在DFC膜片+LPS组表达均高于PDLC膜片+LPS组。因此，DFC膜片比PDLC膜片在炎症环境下更有利于形成牙周新附着和完整的牙周组织结构。2. LPS可能通过TLR4/NF-κB通路抑制了PDLC膜片成骨向分化。DFC膜片在炎症微环境下表达低水平的TLR4，削弱了LPS对DFC膜片成骨向分化抑制。但TLRs及下游信号通路在两种细胞膜片中的调控作用仍需进一步的研究。 3.同种异体比格犬DFC膜片和PDLC膜片植入比格犬牙周炎原位缺损区，均能够在缺损区再生形成新的牙周组织。但DFC膜片组新生骨高度明显高于PDLC膜片，形成牙周再附着的能力更强。.综上所述，炎症刺激明显削弱了PDLC膜片的成骨向分化优势，却促进了DFC膜片成骨向分化并对粘附相关基因的表达无影响。DFC膜片和PDLC膜片在比格犬牙周炎原位缺损区均能够再生形成新的牙周组织，DFC膜片形成再附着能力更强。