BMSC外泌体通过miR-1207-5p调控巨噬细胞NF-κB/TNF-α信号环路调控BMSC分化的机制研究

Spinal cord injury (SCI) is a highly disabling central nervous system injury that still lacks effective treatment. In recent years, Bone marrow stromal cell (BMSC) have the ability to differentiate into nerve cells and can regulate macrophages-secreted TNF-α, which plays an important role in SCI repair. Our previous work indicates that TNF-α can inhibit the differentiation of BMSC and delay the recovery of SCI. However, it is unclear whether BMSC affects its own differentiation and participates in SCI repair by reducing the secretion of TNF-α. Sequence analysis indicated that miR-1207-5p, which is highly expressed in BMSC exosomes, may be targeted regulate the expression of MYD88. MYD88 is a key protein in the NF-κB pathway that regulates TNF-α secretion. Therefore, we hypothesized that miR-1207-5p in BMSC exosomes inhibits NF-κB pathway by targeting MYD88, which led to inhibit the secretion of TNF-α from macrophages, and finally reduce the differentiation inhibition of BMSCs into neural cells by TNF-α, so as to achieve the purpose of SCI repair. This study will validate these hypotheses at the cellular and animal levels, and the results will provide novel idea for cell transplantation therapy in SCI.

脊髓损伤（SCI）是一种高致残率的中枢神经系统创伤，目前仍缺乏有效的治疗手段。骨髓间充质干细胞（BMSC）因具有分化神经细胞的能力，可调控降低巨噬细胞TNF-α的分泌，在SCI修复中起重要作用。我们的前期工作表明TNF-α能抑制BMSC的分化，延缓SCI的恢复。然而BMSC是否通过调控降低TNF-α的分泌从而影响自身分化参与SCI修复尚不清楚。分析表明BMSC外泌体中高表达的miR-1207-5p或可靶向调控TNF-α分泌的重要NF-κB途径中的关键蛋白MYD88的表达。由此我们推测：BMSC外泌体中的miR-1207-5p通过靶向作用MYD88抑制NF-κB通路从而抑制巨噬细胞的TNF-α的分泌，进而减少TNF-α对BMSC向神经细胞分化的抑制作用，从而达到脊髓损伤修复的目的。本研究通过细胞和动物水平验证上述假设，其结果将为SCI的细胞移植治疗提供新的提示。

中枢神经系统的可塑性差，再生能力弱，神经功能的恢复受到很大限制。作为一种细胞间通讯介质，外泌体在治疗脊髓损伤方面独具优势，尤其是来源于BMSCs的外泌体。我们首先在细胞水平验证了mBMSCs-Exo对巨噬细胞TNF-α分泌的影响，再通过mBMSCs的miR-151-5p基因过表达或敲低表达，获得富含miR-151-5p或痕量miR-151-5p的mBMSCs-Exo，用这些外泌体处理M1型巨噬细胞，建立其与TNF-α表达量的联系；利用双荧光素酶报告基因技术，验证了miR-151-5p与MYD88的直接靶向作用，并通过检测NF-κB信号通路蛋白的活性水平，阻断其中的关键蛋白MYD88，说明mBMSCs-Exo中miR-151-5p对巨噬细胞TNF-α表达的调控是通过NF-κB/TNF-α信号通路实现的；使用外泌体处理过的巨噬细胞的上清液去培养mBMSCs，观察细胞的分化状况，进而说明miR-151-5p调控巨噬细胞TNF-α的分泌对mBMSCs分化的影响；最后，我们在动物水平验证细胞水平获得的结论，建立小鼠SCI模型，尾静脉注射不同miR-151-5p量的MBMSCS-Exo，通过评估造模前后运动功能、体重变化、并检测NF-κB信号通路蛋白的活性水平、检测神经细胞分化情况，进一步验证mBMSCs-Exo通过miR-151-5p调节巨噬细胞NF-κB通路影响TNF-α的分泌，从而影响BMSC细胞的分化。最终验证了mBMSCs-Exo通过miR-151-5p调控巨噬细胞NF-κB/TNF-α信号影响mBMSCs分化的完整环路调控机制，揭示了BMSC来源的外泌体未来可能会成为一种有前途的脊髓损伤的治疗方法。