SHED聚合体外泌体转运miR-26a介导相关级联通路诱导血管形成参与全牙髓再生的作用机制研究

We have previously established a method of regenerating full-length dental pulp based on SHED aggregate (SA), and have successfully carried out clinical trial in the patients with pulp necrosis of immature incisors after traumatic dental injuries. Some of the results were newly published in Science Translational Medicine. However, previous studies found that the above methods are not ideal for mature teeth. Our study found that SA-exosomes could play a major role in the angiogenesis during full-length dental pulp regeneration, in which mir-26a has risen observably. Base on the previous researches, we infer that exosomes could transfer mir-26a to mediate the PTEN/PI3K/AKT cascade effect on the angiogenesis during full-length dental pulp regeneration, but the specific regulation mechanism is still not clear. So in this study, we want to firstly established the model of full-length dental pulp regeneration in mature teeth based on SA transplantation, and secondly to observe the endocytosis patterning of SA-exosomes in implant cell using the specific tracer technique, and then to test the effect of miR-26a on angiogenesis using MicroRNA intervention methods, and further to analyze the regulation mechanism of miR-26a-mediated cascades, finally to evaluate the regeneration efficiency of modified SA transplantation. We wish to elucidate the regulation mechanism that exosomes transfer mir-26a to mediate the related cascade effect on the angiogenesis during full-length dental pulp regeneration, and to establish the more efficient method of full-length dental pulp regeneration for the endodontic diseases of mature teeth.

课题组首次建立基于乳牙牙髓干细胞聚合体（SA）移植的年轻恒牙全牙髓再生方法，成果发表于Science Translational Medicine，但发现对于成熟恒牙的疗效不甚理想。前期发现SA分泌的外泌体（SA-exosomes）可以显著提升牙髓再生的效能和血管形成量，而其中的miR-26a含量明显增高。基于以往研究基础，我们推测外泌体转运miR-26a介导PTEN/PI3K/AKT级联通路诱导血管形成促进全牙髓再生，但其具体机制尚不清楚。故本项目拟采用外泌体示踪、MicroRNA调控等方法，建立成熟恒牙全牙髓再生模型，观察SA-exosomes的作用模式，检测miR-26a对血管形成的影响，分析miR-26a调控植体细胞分化的分子机制，评估优化聚合体用于牙髓再生的功效，以期阐明SA-exosomes转运miR-26a促进血管形成的作用机理，建立更高效的成熟恒牙全牙髓再生治疗方法。

课题组前期利用自体乳牙牙髓干细胞聚合体实现了年轻恒牙牙髓的功能性再生，但是其再生的机制尚不清楚。随后在成熟恒牙再生模型中发现，再生组织的再生速率以及形态结构成熟性等方面均较年轻恒牙差，其中新生血管形成的数量差异尤为显著，说明血运系统形成对牙髓组织再生至关重要。因此明确SHED聚合体用于牙髓再生的血管形成机制以及提升SHED聚合体的成牙分化效能是牙髓再生研究的重要目标。近年研究发现来源于间充质干细胞的外泌体（exosome）不仅能通过其旁分泌功能维持干细胞功能和组织稳态；此外可以作为载体通过全身循环方式，转运信号分子如蛋白质、DNA、 mRNA、 microRNA、脂质、细胞器等，影响靶细胞的命运。那么乳牙牙髓干细胞聚合体外泌体(SA-Exo)是否可以诱导血管形成进而促进牙髓再生，有待深入研究。本研究发现1. 微血管系统重建在牙髓再生中具有重要的作用，SA-Exo可通过诱导血管生成而促进牙髓再生。2. SA-Exo可被相关细胞（SHED、HUVEC）内吞，进而诱导其成血管分化，并提升HUVEC的迁移活化能力。3. SA-Exo转运miR-26a介导TGF-β/Smad2/3信号通路，调控SHED 成血管分化及HUVEC的活化动员。4. 过表达miR-26a的SA-Exo能够通过诱导血管形成，提升牙髓再生的效能。本项目初步探索了SA-Exo在牙髓再生中血管形成过程中发挥作用的分子机制，发现SA-Exo转运miR-26a介导相关信号通路调控植体SHED分化和动员活化宿主HUVEC进而促进再生牙髓组织的血运系统重建。为提升牙髓再生效能提供了一种新方法。.