过表达lncRNA HOXD-AS1 的骨髓间充质干细胞外泌体减轻脊髓缺血再灌注损伤的实验研究

Ischemic injury of spinal cords remains a devastating complication with a high incidence after surgery or endovascular repair of thoracoabdominal aneurysms. In the latest studies, stem cells transfer noncoding RNA, such as lncRNAs and miRNAs, via exosomes to alter the gene expression of recipient cells and promote therapeutic response. Exosomes may substitute stem cells as a “cell-free” strategy in tissues repair. MSCs can regulate the expression of a specific gene packaged in exosomes through gene-modification. LncRNA HOXD-AS1 is a novel tumor-related lncRNA, which expresses in the central nervous system. LncRNA HOXD-AS1 promotes proliferation of glioma cells and inhibits their apoptosis. According to bioinformatics-based databases, miRNA-130a and miRNA-330 are indicated to have possible binding sites with lncRNA HOXD-AS1. lncRNA HOXD-AS1 may inhibit miRNA-130a and miRNA-330 as a ceRNA and affect the permeability of blood-brain barrier, neurite outgrowth and neuronal apoptosis via up-regulation of their target proteins expressions. Little is known about functions of exosomes and lncRNAs in the pathology of ischemic spinal cord injury and neuroprotection. In the current grant, exosomes with high level of lncRNA HOXD-AS1 (HOXD-AS1-MSCs-Exo) will be achieved by over-expressing lncRNA HOXD-AS1 in MSCs. The neuroprotection of HOXD-AS1-MSCs-Exo will be evaluated using cultured spinal cord neurons suffering from oxygen glucose deprivation and a rat model of spinal cord ischemia. The possible molecular mechanisms in such a neuroprotection will also be investigated. The final objective is to explore a novel protective strategy on spinal cords with MSCs-Exo as the vector and lncRNA HOXD-AS1 as the therapeutic target.

缺血性脊髓损伤是胸腹主动脉瘤外科和介入治疗的严重并发症。干细胞通过外泌体转运lncRNA, miRNA等，改变靶细胞基因表达并产生修复作用。外泌体可能成为替代干细胞的无细胞疗法。基因修饰MSCs可以改变外泌体中特定基因的表达。LncRNA HOXD-AS1是新的肿瘤相关lncRNA，在神经组织中表达并促进胶质瘤细胞增殖，抑制凋亡。lncRNA HOXD-AS1可能作为ceRNA调控miRNA-130a和miRNA-330，并通过上调其靶蛋白表达影响血脑屏障通透性、神经突生长和神经元凋亡等。目前关于外泌体和lncRNA在脊髓保护中的研究甚少。本课题利用过表达lncRNA HOXD-AS1的MSCs 获得高表达lncRNA HOXD-AS1的外泌体，在培养脊髓神经元和大鼠脊髓缺血模型中探讨其神经保护作用及机制，旨在探索一种以外泌体为载体，lncRNA HOXD-AS1为分子靶点的脊髓保护策略。

脊髓缺血再灌注损伤所致的轻瘫或截瘫是外科或介入治疗胸腹主动脉瘤严重的术后并发症之一。本研究以非编码RNA(ncRNAs)，包括lncRNA、miRNA等作为分子靶点，利用骨髓间充质干细胞外泌体作为载体，探索一种减轻脊髓缺血再灌注损伤的神经保护策略。建立阻断大鼠降主动脉的脊髓缺血再灌注模型和鞘内注射进行基因转染和外泌体输入的实验方法。构建慢病毒或腺相关病毒载体进行基因转染。利用转染骨髓间充质干细胞获取特异性基因修饰的的外泌体。利用神经功能评分和组织学检查确定脊髓损伤程度。qRT-PCR检测lncRNA和miRNA的表达水平。Western blot检测蛋白的表达水平。结果表明，脊髓缺血可以导致后肢运动功能障碍，脊髓组织结构破坏以及健存神经元显著减少。基因转染显著上调脊髓组织中lncRNA HOXD-AS1的表达。过表达lncRNA HOXD-AS1显著改善脊髓缺血后的后肢运动功能，并减轻的神经元凋亡和脊髓组织结构的破坏。LncRNA HOXD-AS1作为ceRNA直接结合miR-130a-3p和miR-330-5p，上调脊髓组织中Homeobox A5, Aquaporins 4 M1, MeCP2, E2F8, E2F1, Occludin和p-Akt等的表达，继而促进神经突生长、减轻神经组织水肿、促进神经元存活、抑制神经元凋亡；过表达lncRNA CRNDE也可以显著减轻脊髓缺血再灌注损伤，作用机制可能是lncRNA CRNDE直接结合miR-181a-5p，进而上调Sirt1的表达。Sirt1的下游靶点POXO1、HIF-1α和PHD2等也参与了神经保护过程。miR-455-5p基因转染骨髓间充质干细胞可以显著上调其外泌体中miR-455-5p的含量。过表达miR-455-5p的外泌体能够显著上调脊髓组织中miR-455-5p的表达，靶向抑制Nogo-A的表达，进而通过抑制神经元凋亡、促进神经元自噬等介导产生减轻脊髓缺血再灌注损伤的神经保护作用。利用大鼠深低温停循环(DHCA)模型，证实骨髓间充质干细胞外泌体在DHCA术后脑损伤中具有显著的治疗作用，机制可能与其抑制炎症反应及促进血管神经再生相关。过表达miR-214的外泌体通过靶向抑制PTEN、Bim和Bax，进而抑制神经元凋亡等进一步提外泌体的神经保护作用。