LDH-NT3@外泌体激活内源性神经干细胞修复脊髓损伤及其机制研究

Spinal cord injury (SCI) repair is a worldwide unresolved problem. The most difficult issues are neural regeneration and neural circuit reconstruction in lesion sites. It is reported that after injury, the survival rate and effective neural differentiation of endogenous neural stem cells (eNSCs) is pretty low. At present, many biomaterials and stem cell secretions are designed to improve the microenvironment, activate eNSCs, achieve neural regeneration and neural circuit reconstruction, and then to repair SCI. This project will be based on the previous research on maintaining stem cell stemness and promoting neural regeneration using layered double hydroxide (LDH). Mesenchyma stem cell (MSC) exosome and neurotrophic factor NT-3 are introduced here to construct LDH-NT3@exosome. The in vivo and in vitro models are used to verify the effectiveness and mechanism of LDH-NT3@Exosome on its SCI repair function. The key genes and pathways are identified by RNA-Seq and WGCNA analysis, and 10x Genomics single cell sequencing is used to analyze and verify the subtypes and lineages of newborn cells in the injured area. Through the project, we can obtain a novel bio-nanocomposite for SCI repaire, reveal the molecular mechanisms of eNSCs activation and how they participate in repair process, and provide theory basis for activating eNSCs to regenerate neuron and reconstruct neural circuit.

脊髓损伤修复是一个世界难题，其难点在于神经再生和环路重建。研究表明，脊髓损伤后内源性神经干细胞(Endogenous Neural Stem Cells, eNSCs)存活率低且不能有效分化，生物材料和干细胞旁分泌技术可激活eNSCs并调控微环境，从而促进神经再生和环路重建以修复脊髓损伤。本项目将在前期对层状双氢氧化物（LDH）维持干细胞干性和促进神经再生的研究基础上，采用LDH搭载神经营养因子NT-3和间充质干细胞外泌体，构建LDH-NT3@外泌体纳米复合体，通过体内外实验分析其激活eNSCs修复脊髓损伤的有效性和相关机制，利用RNA-Seq联合WGCNA分析找出关键调节因子，采用10x Genomics单细胞测序解析新生细胞亚型。项目的实施将获得一种新型脊髓损伤修复材料，揭示新材料干预下eNSCs参与修复的分子机制，为激活eNSCs重建神经环路提供理论依据。

脊髓损伤修复是一个世界难题，其难点在于神经再生和环路重建。研究表明，脊髓损伤后内源性神经干细胞(Endogenous Neural Stem Cells, eNSCs)存活率低且不能有效分化，生物材料和干细胞旁分泌技术可激活eNSCs并调控微环境，从而促进神经再生和环路重建以修复脊髓损伤。本项目采用LDH搭载神经营养因子NT-3得到LDH/NT-3复合生物活性材料，将间充质干细胞来源的外泌体与纤维蛋白胶复合，制备了生物相容性良好的Gel-Exo复合支架体系。通过体内外实验分析两者对脊髓损伤修复的有效性和相关机制。结果证实（1）LDH纳米材料可显著促进神经干细胞（NSCs）迁移、神经分化、激活L-Ca2+通道并诱导动作电位的产生。将LDH负载神经营养因子NT3后形成纳米复合体系（LDH-NT3）移植于脊髓损伤（SCI）小鼠的损伤区域，在损伤区可见新生BrdU+内源性NSCs和功能神经元，显著提高脊髓损伤（SCI）小鼠行为学和电生理评价。LDH材料自身即具有促神经再生作用，并且LDH-NT3对脊髓损伤（SCI）小鼠的脊髓损伤修复效果比LDH组有进一步提高。层状双氢氧化物（LDH）可构建适合脊髓损伤（SCI）修复的免疫微环境，并可负载各类神经营养因子，为脊髓损伤治疗提供生物材料免疫调控新策略。（2）与此同时，经Gel-Exo治疗的小鼠，其行为学评分由0.6分提高至2.4分，较未治疗损伤组有显著改善。同时，与未治疗损伤组相比较，Gel-Exo治疗显著增强了SCI后运动诱发电位（MEP）的振幅（从3.23 μV提高至25.76 μV），并上调了损伤区神经元标志物的表达水平。进一步地，转录组测序结果发现VGF是Gel-Exo促进SCI修复的关键调控因子。制备的Gel-Exo支架材料，其生物相容性较好，有望应用于临床。同时，发现了外泌体中的有效成分VGF，进而揭示了Gel-Exo通过VGF介导的少突胶质细胞再生促进SCI修复的内在机制，为SCI治疗提供了新思路和新靶点。项目的实施获得了新型脊髓损伤修复材料，揭示新材料干预下参与脊髓损伤修复的分子机制，为重建神经环路提供理论依据。