MSCs源exosome促进小胶质细胞M2极化修复脊髓缺血/再灌注损伤的作用及机制

Spinal cord ischemia/reperfusion injury (SCII) often aggravates the spinal cord damage, and its physiopathologic mechanism remains unclear. In pre-experiment, we found that human umbilical cord mesenchymal stem cell-derived exosome (hucMSC-Ex) can promote the proliferation of nerve cells, reduce the cell damage and promote the repair of spinal cord injury in rats, which may be related to the promotion of M2 polarization and the inhibition of M1 polarization. Further studies showed that protective effects and the M2 polarization were decreased after inhibition of CD73-expression on the surface of hucMSC-Ex. Therefore, we propose that hucMSC-Ex may regulate the balance of M1/M2 polarization in microglia by upgrading the expression of CD73 via the activation of adenosine/PI3K/Akt signaling pathway, and then play a protective role in SCII. This study adopts the in vitro model of hypoxia nerve cells and the SCII in vivo model of rats by non-invasive artery clamp closed on aortic arch with systematical hypotension, respectively. From the overall, the cellular and molecular level, we will confirm the repair effects and the mechanism of hucMSC-Ex in SCII, and verify the molecular pathways and the key functional molecule. It will provide new methods and ideas so as to guide the clinical prevention and treatment of SCII, and it has important significance and prospect for clinical application.

脊髓缺血再灌注损伤（SCII）常造成脊髓损伤加重，其确切机制尚不清楚。我们前期的研究发现，脐带间充质干细胞源外泌体（hucMSC-Ex）促进神经细胞增殖、减轻损伤、加速大鼠脊髓损伤的修复，还发现它们促进小胶质细胞M2极化和抑制M1极化。进一步研究显示，抑制hucMSC-Ex表面CD73表达后，hucMSC-Ex对上述的保护和极化作用减弱。因此，我们提出hucMSC-Ex可能通过CD73激活腺苷/PI3K/Akt信号通路调节小胶质细胞M1/M2的平衡，在SCII中发挥保护作用。为此，本研究将采用缺氧神经细胞模型和无创动脉夹夹闭主动脉弓合并系统低血压的方法建立大鼠SCII模型，分别从体内和体外实验，从整体、细胞和分子水平上探讨hucMSC-Ex在SCII中修复的效果和机制，将阐明其发挥功能效应的途径及关键分子，从而为指导临床SCII的防治提供新的方法和思路，具有重要意义和临床应用前景。

目的：从整体、细胞和分子水平上探讨，CD73修饰的人脐带血间充质干细胞源外泌体（exosomes derived from human umbilical cord mesenchymal stem cells, hucMSC-Exs）通过调节小胶质细胞M1/M2的平衡，在脊髓损伤中发挥神经抗炎作用，并进一步阐明其神经保护的机制。.方法：通过慢病毒载体诱导人脐带血间充质干细胞上调CD73的表达，并分离得到CD73+hucMSC-EXs。采用透射电镜、纳米颗粒跟踪分析（NTA）、Western Blot法确认外泌体，并检测ATP水解得到腺苷的能力。体外，采用BV2小鼠小胶质细胞，通过免疫荧光法检测在鉴定了不同条件下小胶质细胞M1/M2亚群的极化情况。通过分别给予腺苷A2aR和A2bR抑制剂SCH58261和MRS1706，以及shRNA相应A2bR水平的敲低，检测小胶质细胞M1/M2极化相应作用。动物实验中，取 ICR小鼠以Allen’s重物坠落法建立脊髓损伤模型，BBB评分评估小鼠运动功能，检测脑脊液ATP水解程度，HE染色和Nissl染色进行形态学分析，流式技术检测M2:M1极化水平，Bio-Plex多重细胞因子试剂盒检测23项细胞因子的水平。.结果：本研究中，通过慢病毒载体诱导并分离得到CD73+hucMSC-EXs。体外实验中CD73+hucMSC-EXs降低了ATP的浓度，升高了腺苷的水平，巨噬细胞/小胶质细胞M1/M2蛋白质标记物表明促进M2极化，抑制M1极化，并且激活了A2bR，促进PKA/cAMP信号通路，升高M1/M2下游mRNA的表达水平。在A2aR和A2bR抑制剂SCH58261和MRS1706以及A2bR shRNA干扰下都证实了CD73+hucMSC-EXs的A2bR/PKA/cAMP靶点作用。在SCI小鼠中，CD73+hucMSC-EXs通过降低脑脊液中ATP的含量，改善M1向M2表型的极化，改善SCI后的功能恢复。同时，促炎细胞因子下调，如TNF-α、IL-1、IL-6，而抗炎细胞因子上调，如IL-10、IL-4。.结论：CD73+hucMSC-EXs通过促进巨噬细胞/小胶质细胞M2:M1极化。激活下游A2bR/PKA/cAMP信号通路，减轻脊髓损伤后的炎症反应，在脊髓损伤的临床治疗中具有良好的应用前景。