人脐带间充质干细胞来源的外泌体介导miR-21调控血管新生及其在骨愈合中的作用机制研究

Nowadays, the mechanism of the therapeutic activity of Mesenchymal Stem Cells (MSCs) in bone healing remains unclear. In spite of the capacity to differentiate, evidence is mounting that much of the therapeutic activity is due to the paracrine effect of the cells. Recent studies have proposed that exosome plays an important role in intercellular communication and function regulation. In previous study, we first discovered that exosome delivered from Human Umbilical Cord Mesenchymal Stem Cells (uMSC-Exo) could, similarly with MSCs, enhanced bone healing by promoting angiogenesis, but the main substance and mechanism of this process needs further investigation. With the high-throughput sequencing and bioinformatics analysis, we preliminary identified that the miR-21 from uMSC-Exo may closely related to angiogenesis. The purpose of this study is to regulate the content of miR-21 in exosome, and to investigate the intercellular transfer of miR-21 by fluorescence in situ hybridization and its effects and molecule mechanism in critical links of angiogenesis. Furthermore, an ischemic non-union model will be utilized to explore its therapeutic value. This study not only enriches the mechanism of the therapeutic activity of MSCs in bone healing, but also provides new theoretical basis and research direction for the molecule therapy of non-union of fracture.

当前人们对间充质干细胞（MSCs）促进骨愈合的机制仍缺乏全面了解。相比较自身分化替代，MSCs的旁分泌作用可能更为重要。新近研究证实，细胞旁分泌的外泌体承担着信息传递及功能调节的重要作用。我们在前期工作中发现人脐带MSCs来源的外泌体（uMSC-Exo）通过增强血管新生，起到“干细胞样”的促进骨愈合作用，而参与这个过程的主要物质和作用机制仍待进一步研究。利用高通量测序及生物信息学分析，我们发现外泌体中的miR-21可能起到关键作用。在此基础上，本课题拟构建不同miR-21水平的外泌体，观察miR-21经外泌体向血管新生细胞转运情况，探索uMSC-Exo介导miR-21对血管新生关键环节的作用和分子机制，并在动物模型中检验 “uMSC-Exo—miR-21—血管新生”在预防骨不连中的作用。本项目的实施，不仅可以丰富MSCs促进骨愈合的机制研究，更为骨不连的分子治疗提供新的理论依据和研究方向。

当前人们对间充质干细胞（MSCs）促进骨愈合的机制仍缺乏全面了解。相比较自身分化替代，MSCs的旁分泌作用可能更为重要。新近研究证实，细胞旁分泌的外泌体承担着信息传递及功能调节的重要作用。本项目一方面发现人脐带MSCs来源的外泌体（uMSC-Exo）可以通过增强血管新生，起到“干细胞样”的促进骨愈合作用。通过进一步的高通量测序及生物信息学分析，发现外泌体中的miR-21丰度突出且与血管化作用有密切关系。在此基础上，我们成功拟构建不同miR-21水平的脐带间充质干细胞外泌体，证实了miR-21经脐带间充质干细胞外泌体向靶细胞血管内皮细胞转运情况，验证了uMSC-Exo介导miR-21对血管新生关键环节，包括促进血管内皮细胞增殖、分化、管腔形成等的作用和VEGF、HIF-1a等血管化相关因子表达上调，并通过通过生物信息学研究发现PTEN的3′UTR 存在miR-21的作用位点，过表达miR-21可抑制PETN的3′UTR 荧光素酶的表达活性。通过转染miR-21的agomir及antagomir，在HUVECs中过表达或抑制miR-21，发现调控miR-21的变化可以在mRNA和蛋白水平影响PTEN和其下游靶点VEGFA、HIF-1α的表达以及PI3K/AKT的磷酸化水平，最终明确了其下游的具体信号通路。最终在动物模型中初步验证了 “uMSC-Exo—miR-21—血管新生”在骨不连中的潜在治疗作用。本项目的实施，不仅丰富MSCs促进骨愈合的机制研究，更为骨不连的分子治疗提供新的理论依据和研究方向。