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。

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.