脱落乳牙干细胞外泌体通过miR-27b-3p阻断神经元焦亡的机制研究

The paracrine effect plays a major role in MSCs-based tissue repair, and exosome is the most important component of paracrine, which can penetrate the blood-brain barrier to the injured area directly. Stem cells from exfoliated deciduous teeth（SHED）are ectodermal neural crest-derived adult stem cell population，homologous to central nervous tissue and have strong ability of proliferation and neural differentiation. The proposer previously found that SHED-Exo has is good effect on the neuronal pyroptosis caused by ischemia, but the detailed mechanism is not clear. And the sequencing results revealed that miR-27b-3p is highly expressed in SHED-Exo. It was predicted that miR-27b-3p targeted modulates the transcription factor IKZF2, which binds to the promoter region of NLRP3, a key factor of pyroptosis. Therefore, we proposed a hypothesis that SHED-Exo could be taken up by damaged neurons, targeted modulate IKZF2 through miR-27b-3p, and then down-regulated the expression of NLRP3 and blocked the neuronal pyroptosis. This project intends to elucidate the mechanism of inhibiting the neuronal pyroptosis for SHED-Exo through miR-27b-3p, and lays a foundation for the application of dental stem cells-derived exosome on the nerve injury treatment.

间充质干细胞主要通过旁分泌作用发挥组织修复功能，而外泌体是旁分泌中最主要的成分，能够穿透血脑屏障到达损伤区域促进神经功能恢复。脱落乳牙干细胞（SHED）是外胚层神经嵴来源的成体干细胞群，与中枢神经组织同源，具有很强的增殖和神经向分化能力。申请人前期发现SHED来源的外泌体（SHED-Exo）对缺血导致的神经元焦亡具有抑制作用，但具体机制尚不清楚。进一步研究发现SHED-Exo高表达miR-27b-3p，而miR-27b-3p靶向调控转录因子IKZF2，后者与焦亡关键因子NLRP3启动子区存在结合位点。因此提出假说：SHED-Exo被受损神经元胞吞并通过miR-27b-3p靶向调控IKZF2，进而下调NLRP3表达，阻断神经元焦亡。本项目旨在解析SHED-Exo通过miR-27b-3p阻断神经元焦亡的分子机制，为牙源性干细胞外泌体应用于神经损伤治疗奠定基础。

神经系统疾病多与免疫炎症、氧化应激、神经性毒性等导致的神经元数量减 少和功能损伤密切相关。通常情况下，神经元损伤后不能再生，尽管临床采用综合救治、神经保护及康复期功能重建等手段治疗神经损伤，但效果不佳，尚缺乏有效的治疗方法。近年来，随着干细胞研究的深入，以干细胞为基础的细胞治疗在急、慢性组织损伤、免疫缺陷及癌症治疗方面取得了可喜的研究成果，为神经系统疾病的治疗供了新方向。间充质干细胞主要通过旁分泌作用发挥组织修复功能，外泌体是旁分泌中最主要的成分，能够穿透血脑屏障到达损伤区域促进神经功能恢复。因此，间充质干细胞来源的外泌体作为无细胞制剂有望成为神经系统疾病治疗的新策略。.脱落乳牙干细胞（stem cells from exfoliated deciduous teeth, SHED）是外胚层神经嵴来源的成体干细胞群，与中枢神经组织同源，具有很强的增殖和神经向分化能力。本项目研究发现，SHED及其来源的外泌体（SHED-Exo）能够有效缓解因缺血导致的认知功能障碍，抑制神经元焦亡，恢复脑缺血大鼠海马区神经元的数量及功能，促进神经损伤后的组织修复。课题组进一步应用体内体外实验揭示了SHED通过旁分泌SHED-Exo到达受损神经元周围，被受损神经元胞吞并转运miR-27b-3p，靶向调控焦亡关键因子NLRP3的表达水平，阻断神经元焦亡，促进神经元的损伤修复的分子机制。本研究提出利用脱落乳牙干细胞来源的外泌体具有高度细胞起源依赖性且能穿透血脑屏障的优势，转运生物活性物质，促进神经组织损伤修复的理念，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实验基础和理论依据。