SDF-1/CXCR4轴介导内源性细胞归巢的牙髓再生机制研究

It is now well-documented that stem cells, which have the capability of pulp regeneration, could be obtained from both odontogenic and non-odontogenic tissues, such as adipose tissue or bone marrow. Considering the risk of cells transplantation, endogenous regeneration through cell homing provides a promising approach for pulp regeneration. The chemokine stromal cell-derived factor-1 (SDF-1) which is the only ligand for the CXC chemokine receptor 4 has been considered as the most predominant stem cells homing factor. SDF-1/CXCR4 axis plays a pivotal role in various tissue/organ regeneration by mobilizing and homing stem/progenitor cells to injured site, including vascularization, neurogenesis and osteogenesis. Unlike other tissue regeneration, the regenerated pulp tissue is encapsulated in the highly mineralized dentin wall and merely contacts to the periodontal tissue through the apical formation. Based on the current clinical case reports of managing the immature permanent tooth by the dental pulp revascularization therapy strategy, the endogenous cells will instantly migrate into the root canal with the blood after the mechanical stimulate. However, the biological activity and the amount of the cells which migrated into the root canal contribute to pulp reconstruction and the mechanism of sustainable recruitment cells is remain unknown. The project investigate the releasing pattern of SDF-1 activated by periapical tissue injury, the dynamic process of recruiting endogenous stem cells into root canal and the biological activity of homing CXCR4+ cells by using tracking technique based on overexpressing CXCR4-bone marrow mesenchymal stem cells(CXCR4-BMMSCs) and RNAi-BMMSCs in both vivo and vitro. We determinate for exploring the mechanism of SDF-1/CXCR4 axis mediating dental pulp regeneration, which is expected to provide the scientific evidences for optimization clinical therapy strategy.

人体内存在多种具有牙髓再生功能的干细胞，包括牙源性和非牙源性的干细胞，有效动员体内干细胞再生功能的内源性牙髓再生途径可以避免干细胞移植导致的风险。SDF-1被认为是最重要的干细胞归巢因子，它与 CXCR4特异性结合后形成的SDF-1/CXCR4轴在多种组织/器官的再生中发挥作用，主要通过介导干/祖细胞归巢及参与血管、神经和骨的形成。目前临床上实施的牙髓血管化再生治疗，除了已知体内的细胞在机械刺激触发后瞬间随血流进入根管腔内外，对进入根管腔内机体细胞的生物学活性，持续诱导细胞归巢发生的可能机制仍然未知。本项目采用基因工程技术构建CXCR4高表达 CXCR4+骨髓间充质干细胞系（CXCR4- BMMSCs）及CXCR4基因沉默骨髓间充质干细胞系（RNAi-BMMSCs），通过体内外模型研究SDF-1的释放规律、细胞归巢的机制、CXCR4+归巢细胞的生物学活性，探讨SDF-1/CXCR4生物轴在血管化再生过程中的作用机制，阐述内源性牙髓再生过程中细胞归巢的生物学过程及影响因素，为优化临床治疗策略提提供理论依据

重建功能性牙髓是牙髓病和根尖周病治疗的理想目标，内源性干细胞归巢形式的牙髓再生具有临床易推广、风险小等优点。本研究旨在通过一系列体外、体内研究，探讨SDF-1/CXCR4轴募集内源性干细胞归巢参与牙髓修复再生的作用机制。通过项目的实施，本研究进行了：1、评价根尖周局部损伤信号所诱发的血清和根管内SDF-1的表达及骨髓中CXCR4+细胞的变化情况；2、体外评价SDF-1对BMSCs的趋化作用；3、构建CXCR4高表达和沉默的BMSCs，采用示踪技术体内评价SDF-1/CXCR4轴在牙髓再生性治疗过程中募集系统干细胞归巢参与再生的生物学作用；4、RNA-seq评价刺激不同的再生性牙髓治疗措施新生组织mRNA的差异表达。通过该项目的研究，获得了具有临床指导价值的科学结果：1、牙髓血管化再生治疗通过根尖损伤可以激发血清中的SDF-1分别在12小时内和4d时出现高峰，骨髓中的血管扩张，扩张的血管中聚集了大量的CXCR4阳性表达细胞；2、体内及体外的研究结果均表明，SDF-1可以趋化BMSCs细胞在体内、体外的迁移，无论是损伤引起的还是根管内植入SDF-1，局部SDF-1浓度的升高，均可趋化体内系统的BMSCs归巢到达根尖周组织和根管内，我们发现该作用可能存在浓度依赖性，当在动物体内双侧同时进行不同方式的牙髓再生处理时，系统BMSCs明显聚集到SDF-1浓度较高的一侧，证实归巢的BMSCs参与了牙髓的修复与再生。同时，我们也观察到，CXCR4表达与沉默的BMSCs在体内主要脏器中的分布也不同，沉默的细胞主要从脾脏和肾脏中清除，而不到达根尖周和根管中。这一系列的结果证实SDF-1/CXCR4轴在募集内源性干细胞归巢参与牙髓组织再生修复中具有重要的意义。RNA-seq检测提示成骨相关基因在牙髓血管化再生治疗和根管内植入SDF-1两种处理中起关键调控作用，而成牙本质向、神经向基因下调表达，这可能是再生组织尚未能完全达到理想牙髓组织结构和功能的表现之一。