人脐带间充质干细胞外泌体通过microRNA-34c-5p/RUNX2轴调控骨再生的作用研究

Role of human umbilical cord mesenchymal stem cell-derived exosomes in regulating bone regeneration via the microRNA-34c-5p/RUNX2 axis

摘要:

目的 探讨人脐带间充质干细胞来源的外泌体（hUC-MSC-Exos）通过microRNA-34c-5p（miR-34c-5p/RUNX2信号轴调控成骨分化的分子机制，并评估硫酸软骨素水凝胶递送系统的治疗效果。方法 采用差速离心法分离hUC-MSC-Exos，并通过Western blotting检测外泌体标志物CD63及CD81。构建miR-34c-5p过表达载体并合成其抑制剂，采用双荧光素酶报告基因系统验证miR-34c-5p与RUNX2的3'-非翻译区（3'-UTR）的靶向结合关系。制备硫酸软骨素水凝胶负载的外泌体复合物（Exos@Gel），复制SD大鼠胫骨缺损模型，通过微型计算机断层扫描和组织学分析评估骨修复效果。结果 Western blotting检测结果证实成功分离hUC-MSC-Exos。与细胞裂解液相比，hUC-MSC-Exos中miR-34c-5p表达水平显著升高（P <0.05）。双荧光素酶报告结果显示，miR-34c-5p可直接结合RUNX2的3'-UTR，并使其相对荧光素酶活性显著降低（P <0.05）。功能实验结果显示，过表达miR-34c-5p可抑制RUNX2蛋白表达水平（P <0.05），而敲低miR-34c-5p则促进RUNX2表达水平（P <0.05）。动物实验显示，水凝胶复合外泌体组的骨矿物质含量和新生骨体积均高于水凝胶治疗组（P <0.05）。结论 hUC-MSC-Exos通过递送miR-34c-5p抑制RUNX2表达，从而促进骨再生。结合RUNX2在成骨分化中的“双相调控”理论，提示在骨折愈合过程中抑制RUNX2可能促进骨形成。此外，硫酸软骨素水凝胶可进一步增强该策略的治疗效果。

Abstract:

Objective To investigate the molecular mechanism by which human umbilical cord mesenchymal stem cell exosomes (hUC-MSC-Exos) regulate osteogenic differentiation through the miR-34c-5p/Runt-related transcription factor 2 (RUNX2) signaling axis and, to evaluate the therapeutic effect of a chondroitin sulfate hydrogel delivery system.Methods hUC-MSC-Exos were isolated using differential centrifugation, and exosome markers CD63 and CD81 were detected by Western blotting. An miR-34c-5p overexpression vector and an inhibitor were prepared, and their targeting relationship with the RUNX2 3'-untranslated region (UTR) was verified using a dual-luciferase reporter assay. A chondroitin sulfate hydrogel-exosome complex (Exos@Gel) was prepared. A tibial bone defect model was established in Sprague-Dawley (SD) rats, and bone repair efficacy was assessed by micro-computed tomography (Micro-CT) and histological analysis.Results Western blotting confirmed the successful isolation of hUC-MSC-Exos. Compared with cell lysates, miR-34c-5p expression was significantly higher in hUC-MSC-Exos (P < 0.05). The dual-luciferase reporter assay showed that miR-34c-5p directly bound to the RUNX2 3'-UTR, and significantly decreased its relative luciferase activity. Functional experiments demonstrated that overexpression of miR-34c-5p inhibited RUNX2 protein expression (P < 0.05), while knockdown of miR-34c-5p promoted RUNX2 protein expression (P < 0.05). Animal experiments revealed that the Exos@Gel group was significantly superior to the hydrogel-only treatment group in terms of bone mineral content and new bone volume (P < 0.05).Conclusion hUC-MSC-Exos promote bone regeneration by delivering miR-34c-5p, which inhibits RUNX2 expression. Consistent with the established theory of biphasic regulation of RUNX2 in osteogenic differentiation, this finding suggests the possibility that its inhibition during fracture healing could promote bone formation. The chondroitin sulfate hydrogel significantly enhances the efficacy of this therapeutic strategy.