牙囊干细胞旁分泌重塑牙髓炎症微环境的免疫调节机制研究

Bacterial infection triggers inflammatory cascade in dental pulp and the consequent disorder of pulp microenvironment hinders tissue repair and regeneration. Dental follicle stem cells (DFSCs), possessing a stronger immunomodulatory effect than other dental derived mesenchymal stem cells, is capable of modulating the inflammatory microenvironment and promoting injured tissue repair via paracrine pathway. Our previous study revealed that the condition medium (CM) of rat DFSCs contained abundant bioactive factors, which might play critical roles in immunomodulation, angiogenesis and neurotrophy. The CM was shown to have an anti-inflammatory effect on dental pulp immune cells and enhanced the stemness of rat dental papilla cells. Therefore, this project is to investigate whether DFSCs could modulate the immune responses of dental pulp and optimize the inflammatory microenvironment via paracrine pathway, thus promoting the injured pulp repair. Firstly，the immunomodulatory effects of rDFSCs on LPS-induced rat dental pulp cells and macrophages via paracrine pathway, together with the activation of LPS/TLR4 signaling pathways will be examined. Secondly, the influence of rDFSCs paracrine effect on the repair and regeneration capacities of pulpitis-derived dental pulp stem cells and inflamed dental pulp tissue will be assessed. Lastly, the key paracrine factors will be screened by protein array analysis, and the composite medium containing those key anti-inflammatory factors will be applied as an immunotherapeutic agent to experimental pulpitis of rat for direct pulp capping. This study will help reveal the role of DFSCs in dental pulp immune responses and inflammation processes from a new perspective of immunomodulation, and provide experimental evidence of taking advantages of compound bioactive factors to remodel dental pulp inflammatory microenvironment and developing the minimally invasive bio-therapeutic endodontic strategy.

细菌感染诱发炎症级联反应，导致牙髓微环境失衡，拮抗组织修复再生。牙囊干细胞较其他牙源性干细胞具有更强的免疫调节能力，能通过旁分泌效应改善炎症微环境，促进损伤组织的修复。我们前期研究发现大鼠牙囊干细胞的条件培养基富含免疫调节因子、血管生长因子、神经营养因子等，对牙髓免疫细胞具有抑炎作用、并促进牙乳头细胞的干性。那么能否利用牙囊干细胞的旁分泌特性调节损伤牙髓的免疫应答，重塑炎症微环境，促进组织修复，从而为成人损伤牙髓的保存开辟新的治疗途径？本项目拟检测大鼠牙囊干细胞旁分泌对LPS活化的巨噬细胞和牙髓细胞的免疫调节作用以及LPS/TLR4信号通路激活情况，评估其对炎症牙髓干细胞和炎性牙髓修复再生的影响；蛋白质芯片筛选旁分泌的关键效应因子，用于实验性牙髓炎的直接盖髓，揭示牙囊干细胞对牙髓免疫和组织修复的调节作用及分子机制，为利用生物活性因子重塑炎症微环境、建立牙髓微创生物治疗策略提供实验依据。

细菌感染诱发炎症级联反应，导致牙髓微环境失衡，拮抗组织修复再生。牙囊干细胞较其他牙源性干细胞具有更强的免疫调节能力，能通过旁分泌效应改善炎症微环境，促进受损组织修复。前期研究发现大鼠牙囊干细胞条件培养基（condition medium of rat dental follicle stem cells, DFSCs-CM）对牙髓免疫细胞具有抑炎作用，并促进牙乳头细胞的干性。本课题聚焦牙髓微环境免疫调控作用，构建LPS/粪肠球菌诱导的炎症巨噬细胞模型，发现炎症微环境可上调巨噬细胞促炎因子表达水平，抑制其破骨向分化。在此基础上，以大鼠牙囊干细胞旁分泌调节牙髓炎症微环境为切入点，明确rDFSCs-CM可通过介导巨噬细胞极性转变发挥免疫调节作用，并通过高通量蛋白质芯片技术筛选出潜在效应分子。干细胞多能性在损伤牙髓修复中具有重要作用，本项目发现生物钙硅基类材料浸提液、小鼠胚胎成纤维细胞条件培养基、人脐带间充质干细胞改性微环境可调控牙髓细胞多能性，提示可通过调控微环境改善受损牙髓细胞功能。通过构建炎症牙髓细胞模型，明确rDFSCs-CM可通过ERK 1/2 MAPK和NF-κB信号通路发挥对炎症牙髓细胞的免疫调控作用，并上调炎症微环境中牙髓细胞修复潜能。最后，构建大鼠实验性牙髓炎模型，分析受损牙髓炎症因子表达谱并证明rDFSCs-CM直接盖髓可促进受损牙髓修复。研究成果提示rDFSCs-CM有助于改善牙髓炎症微环境，激发牙髓修复潜能，为进一步揭示牙囊干细胞旁分泌对受损牙髓的免疫调节机制以及开展炎症牙髓微创生物治疗策略提供依据。共发表学术论文11篇，SCI收录7篇，培养博士研究生2名，硕士研究生1名。