外泌体局部植入诱导M2型巨噬细胞极化促进前交叉韧带重建术后腱骨愈合

Excessive scar formation affected the quality of tendon-bone healing after anterior cruciate ligament (ACL) reconstruction. The inflammatory reaction mediated by M1-type macrophages was a crucial factor. The literatures showed that Kruppel-like factor 6 (KLF6) played an important role during the proinflammatory process of M1-type macrophages. Meanwhile, the exosomes could induce the proinflammatory M1-type macrophage to convert to M2-type macrophages, which promoted tissue repair. The previous pilot test confirmed that the exosomes derived from bone mesenchymal stem cells (BMSCs) could be phagocytized by macrophages. The exosome-derived miRNA-let7c was highly expressed in it. We also found that the KLF6 gene contained potential miRNA-let7c silencing binding sites participating in the process of macrophages polarization. Therefore, it was supposed that the local implanted exosomes might reprogram proinflammatory M1-type macrophages to convert to M2-type macrophages, inhibit inflammatory reaction and scar formation, and enhance the possible direct healing of tendon-bone after ACL reconstruction. The objective of the project was to confirm that BMSCs-derived exosomes enhanced the direct healing of tendon-bone by inducing macrophage reprogramming in vitro and vivo levels. Furthermore, we investigated the mechanism hypothesis proposed according to the literatures and our previous pilot experiments: the high expression of miRNA-let7c inhibited KLF6, reprogramed macrophages, and finally enhanced tendon-bone healing. The project was expected to enhance tendon-bone healing by expanding the new non-cellular method, which was easy preparation, had better stability and higher bio-safety. The ideas and concept of current research have not been reported at home and abroad.

前交叉韧带(ACL)重建后过多的瘢痕形成影响腱骨愈合质量，M1型巨噬细胞促成的炎症反应是重要影响因素。文献表明，Kruppel样因子6（KLF6）在M1型细胞促炎中作用重要；外泌体可使促炎的M1型细胞极化为促组织修复的M2型巨噬细胞。预试证实，来源于BMSCs的外泌体被巨噬细胞吞噬，外泌体内miR-let7c高表达并与KLF6有着相应沉默结合位点。据此预测，外泌体局部植入可使促炎的M1型细胞重编程为M2型巨噬细胞，抑制炎症反应及瘢痕形成、促进ACL重建后腱骨直接愈合。本项目拟在细胞和整体水平证实BMSCs源性外泌体通过诱导巨噬细胞重编程为M2型促进腱骨直接愈合；探讨外泌体内高表达的miR-let7c抑制KLF6，致巨噬细胞重编程，最终促进腱骨愈合这一综合文献与预试提出的机制假说。本项目有望拓展出易于制备、稳定性好、生物安全性高的促进腱骨愈合的非细胞性方法。本研究思路及内容国内外尚未见报道。

前交叉韧带(ACL)重建后过多的瘢痕形成影响腱骨愈合质量，M1型巨噬细胞促成的炎症反应是重要影响因素。最近的研究结果证实骨髓间充质干细胞来源的外泌体(bone marrow stromal cell-derived exosomes, BMSC-Exos)可用于组织修复。然而，BMSC-Exos能否促进前交叉韧带重建(anterior cruciate ligament reconstruction, ACLR)术后腱骨愈合目前仍不清楚。在本研究中，我们在体内和体外观察了大鼠BMSC-Exos对ACLR术后腱骨愈合的影响及其可能的机制。通过生物信息学筛选大鼠BMSC-Exos中高表达的miRNA，并进行体外验证。在体外观察了过表达miR-23a-3p的BMSC-Exos对诱导M1巨噬细胞向M2极化的影响。然后，通过建立大鼠ACLR模型，将实验分为3组：对照组、BMSC-Exos组和miR-23a-3p过表达的BMSC-Exos（BMSC-Exos mimic）组。并对各组样本进行了生物力学测试、micro-CT分析和组织学染色以对腱骨愈合情况进行分析。生物信息学分析表明，miR-23a-3p在大鼠BMSC-Exos中高表达，并且可以靶向并调节干扰素调节因子1（interferon regulatory factor 1, IRF1）的表达。在体外，与对照组或BMSC-Exos组相比，BMSC-Exos mimic更显着地诱导了M1型巨噬细胞向M2型巨噬细胞的极化。在体内，2周时，BMSC-Exos mimic 组M2型巨噬细胞数量显着增加；在4周、8周时，与对照组或BMSC-Exos组相比，BMSC-Exos mimic组大鼠胫骨和股骨侧骨隧道面积明显减少，移植物与骨的界面宽度明显减少。骨体积/总体积比增加，II型胶原水平增加，机械强度增加。综合研究结果提示BMSC-Exos通过miR-23a-3p促进M1巨噬细胞向M2巨噬细胞极化，减轻ACLR术后腱-骨界面的早期炎症反应，可促进早期腱骨愈合。