整合素α5（Integrin-α5）对人牙髓干细胞成牙本质向分化的调控及作用机制研究

Pulpitis and apical periodontitis are the most common diseases in the current dental clinic. The ideal treatment for infected pulp refers to regenerative endodontics. To achieve this goal, pulp-dentin complex regeneration, the key points of which are odontogenesis of stem cells, must be emphasized. We had previously reported that integrin-α5 (ITGA5) negatively regulates odontogenesis of human dental pulp stem cells (DPSCs). We aim to determine whether ITGA5 can be applied in dental pulp regeneration in this study. First of all, we will test the ITGA5’s knockout effect on odontogenesis of DPSCs. Second, to tackle with the issues of three-dimensional structure formation and blood supply formation and verify the possibility of ITGA5 knockout DPSCs appliance in pulp-dentin complex regereration, two models will be adopted: one is 3D co-culture model of DPSCs, which will be transfected with KO-ITGA5-sgRNA lentivirus, and endothelial precursor cells (EPCs); the other is murine in vivo subcutaneous transplantation of DPSCs/EPCs 3D beads in a human empty root canal. Last but not least, in order to explore the exact signaling network and search for key molecules during the process of odontogenesis of ITGA5 knockout dental pulp stem cells in pulp-dentin complex regeneration, advanced high throughput techniques such as transcriptome and proteome analysis will be performed. Thus this study will provide theory and research basis for the possible appliance of ITGA5 knockout DPSCs in regenerative endodontics.

牙髓炎和根尖周炎是最常见的口腔疾病，对感染牙髓的理想治疗方法为牙髓再生性治疗，即牙髓牙本质复合体再生，其关键在于干细胞的成牙本质向分化。本课题组前期首次报道整合素α5（ITGA5）对牙髓干细胞成牙本质向分化起重要的负性调控作用。为深入探究ITGA5在牙髓再生治疗中的应用价值，本项目拟首先通过体外验证ITGA5基因敲除对人牙髓干细胞成牙本质向分化的调控作用；其次，采用ITGA5基因敲除的人牙髓干细胞与内皮前体细胞3D共培养细胞模型和人牙空根管小鼠体内移植模型，实现三维结构形成和血运构建进行体内验证；最后，为明确其分子机制，通过转录组、蛋白组测序等新近发展的高通量筛选技术绘制ITGA5调控牙髓干细胞成牙本质定向分化的具体信号通路网络并进行验证，寻找在此过程中关键的信号分子，为揭示ITGA5调控牙髓干细胞成牙本质定向分化的分子机制、阐明ITGA5基因敲除用于牙髓再生治疗的可行性提供实验研究基础。

牙髓炎和牙周炎是口腔临床上最常见的疾病，严重的牙髓炎症和牙周炎症可导致导致牙齿缺失及牙列缺损。牙髓干细胞（Dental pulp stem cells， DPSCs） 的成牙本质向分化和牙周膜干细胞（Periodontal ligament stem cells， PDLSCs）的成骨分化对于牙髓组织和牙周膜组织的修复和再生至关重要。本课题组前期发现整合素 α5 （ITGA5）对 DPSCs 的成牙本质向分化有重要的调控作用，但其确切机制并不清；而ITGA5对PDLSCs成骨分化的作用未见报道。本项目构建靶向调控ITGA5病毒载体和合成ITGA5结合环肽，利用CCK8细胞增殖实验、Transwell小室迁移实验验证ITGA5对DPSCs和PDLSCs增殖和迁移的影响，利用体内外实验技术探讨ITGA5对DPSCs成牙本质向分化和对PDLSCs成骨分化的影响并利用蛋白质学技术探讨其中机制。结果发现：1. 使用ITGA5结合环肽可显著抑制DPSCs的增殖能力和迁移能力。ITGA5抑制表达慢病毒和ITGA5过表达慢病毒分别能显著抑制和增强PDLSCs的增殖能力和迁移能力，ITGA5结合环肽可显著增强PDLSCs的增殖能力和迁移能力；2. ITGA5结合环肽在矿化诱导的条件下可促进DPSCs的成牙本质向分化。在矿化诱导的条件下，抑制DPSCs中的ITGA5表达不仅导致FAK、PI3K/AKT、MEK1/2/ERK1/2信号通路关键分子的磷酸化，而且还导致ECM的大量沉积及ECM-受体活性级联放大，进而促进DPSCs的成牙本质向分化；3. ITGA5过表达慢病毒和ITGA5结合环肽在矿化诱导的条件下可促进PDLSCs的成骨分化，ITGA5抑制表达慢病毒则能抑制PDLSCs的成骨分化。在矿化诱导的条件下，激活PDLSCs中的ITGA5表达不仅导致FAK、PI3K/AKT、MEK1/2/ERK1/2信号通路关键分子的磷酸化，而且还导致细胞骨架和细胞周期的改变，进而促进PDLSCs的成骨分化。本实验阐明了靶向调控ITGA5促进DPSCs成牙本质向分化和PDLSCs成骨分化的作用机制，为ITGA5结合环肽GA-CRRETAWAC-GA应用于牙髓病和牙周病的临床治疗提供理论基础。