SCAP外泌体通过Tet2调控Treg细胞功能介导牙髓再生的分子机制

Stem cells from apical papilla (SCAP)-based tissue engineering is a new strategy for the treatment of immature permanent teeth affected with pulpal and periapical disease. Local immune microenvironment plays an important role in tissue regeneration. However, the underlying mechanisms by which donor SCAP modulate recipient cell function in dental pulp regeneration are not fully understood. In our previous studies, Beagle dogs were used to conduct apical periodontitis and perform regenerative endodontic treatment, and it was indicated that Treg was involved in tissue regeneration. Moreover, in vitro SCAP induced conversion of CD4+ T cell to Treg by paracrine effects and in which exosome secretion is a major mechanism. Treg specific hypomethylation phenotype is a key factor for stability of immune-suppressing function. We further found that exosomes derived from SCAP expressed higher level of Tet2. Therefore, we hypothesize that SCAP-derived exosomes transfer Tet2 to T cell, then regulate Treg function by Tet2-mediated demethylation. Consequently SCAP-derived exosomes are able to reprogram the local immune microenvironment and improve pulp regeneration. This project will not only indicate local immune microenvironment as a new target of tissue regeneration, but also provide theoretical and experimental evidence for the application of SCAP-derived exosomes in dental pulp regeneration.

以根尖牙乳头干细胞（SCAP）为基础的组织工程技术是年轻恒牙牙髓及根尖周病治疗的新策略，局部免疫微环境在组织再生中起关键作用，但SCAP调控宿主细胞功能介导牙髓再生的机制尚不清楚。本课题组前期应用比格犬动物模型研究发现调节性T细胞（Treg）在牙髓再生中发挥重要作用，细胞共培养发现SCAP通过旁分泌作用诱导CD4+T细胞向Treg转化，且外泌体是SCAP旁分泌的重要途径。特异性低甲基化的表观遗传特征是Treg免疫抑制功能稳定发挥的关键；课题组同时发现SCAP外泌体高表达去甲基化调控酶Tet2。因此，本研究拟阐明SCAP来源外泌体传递Tet2至T细胞并被其再利用，通过Tet2介导Treg关键基因的去甲基化修饰，促进Treg转化并获得稳定免疫抑制功能，重塑免疫微环境，促进牙髓再生。由此提出以局部免疫微环境作为组织再生新靶点，并为SCAP来源外泌体应用于牙髓再生提供理论和实验依据。

年轻恒牙牙根的继续发育依赖于有活力的牙髓和根尖牙乳头组织。如果年轻恒牙罹患牙髓及根尖周病，将影响牙根的继续发育，造成牙根薄弱易折裂，冠根比例不协调，甚至引起牙齿过早脱落，严重影响患儿的咀嚼功能、生长发育及心理健康。传统的根尖诱导成形术和根尖屏障术不能使牙根继续发育达到应有的长度和根管壁厚度，并且根管内仅为充填材料，失去牙髓的感觉功能和免疫防御反应。以干细胞为基础的组织工程技术为年轻恒牙牙髓及根尖周病的治疗提供了新的方向，但临床上直接应用干细胞仍存在诸多的困难。外泌体是真核细胞分泌到细胞外的一种膜性微囊泡，携带亲代细胞的特征性生物学信息，并且性质稳定，易于储存，无伦理学问题。因此，干细胞外泌体作为无细胞制剂有望成为于年轻恒牙牙髓再生治疗的新策略。.根尖牙乳头干细胞来源于牙根生长发育的核心组织牙乳头，能够分化为成牙本质细胞，是牙髓再生治疗中极具应用前景的干细胞源。本项目研究发现，应用根尖牙乳头干细胞来源的外泌体（exosome derived from stem cells from apical papilla, SCAP-Exo）能够促进比格犬罹患根尖周炎的年轻恒牙牙髓再生，达到接近生理性的牙根发育，还可有效缓解大鼠早期牙髓炎症，促进牙髓损伤后的组织修复。课题组进一步应用体内体外实验揭示了SCAP-Exo通过Tet2介导DNA去甲基化修饰，促进调节性T细胞（regulatory T cell, Treg）转化并获得稳定免疫抑制功能，通过上调局部组织中Treg/CD4+ T细胞比率及IL-10水平、下调IL-17水平，重塑局部免疫微环境，促进组织再生。本研究提出利用根尖牙乳头干细胞来源的外泌体的免疫调节性能，针对局部免疫微环境调控，促进牙髓组织再生的理念，为将牙源性干细胞外泌体作为无细胞制剂应用于组织再生的临床转化提供重要理论和实验依据。