中国组织工程研究

中国组织工程研究 ›› 2024, Vol. 28 ›› Issue (7): 985-992.doi: 10.12307/2024.109

• 骨髓干细胞 bone marrow stem cells •    下一篇

1%富血小板血浆联合骨髓间充质干细胞促进周围神经损伤的修复

冯睿钦1，2，3，韩  娜1，2，3，张  蒙1，2，3，谷馨怡1，2，3，张丰识1，2，3   

  1. 1北京大学人民医院创伤骨科，北京市   100044；2创伤与神经再生教育部重点实验室，北京市   100044；3国家创伤医学中心，北京市   101100
  • 收稿日期:2023-02-01 接受日期:2023-03-09 出版日期:2024-03-08 发布日期:2023-07-15
  • 通讯作者: 韩娜，博士，研究员，北京大学人民医院创伤骨科，北京市 100044；创伤与神经再生教育部重点实验室，北京市 100044；国家创伤医学中心，北京市 101100
  • 作者简介:冯睿钦，男，1997年生，福建省邵武市人，汉族，北京大学医学部在读硕士，医师，主要从事创伤与周围神经再生相关研究。
  • 基金资助:
    北京市自然科学基金(7222198)，项目负责人：韩娜

Combination of 1% platelet-rich plasma and bone marrow mesenchymal stem cells improves the recovery of peripheral nerve injury

Feng Ruiqin1, 2, 3, Han Na1, 2, 3, Zhang Meng1, 2, 3, Gu Xinyi1, 2, 3, Zhang Fengshi1, 2, 3   

  1. 1Department of Orthopedics and Trauma, Peking University People’s Hospital, Beijing 100044, China; 2Key Laboratory of Trauma and Neural Regeneration, Ministry of Education, Beijing 100044, China; 3National Center for Trauma Medicine, Beijing 101100, China
  • Received:2023-02-01 Accepted:2023-03-09 Online:2024-03-08 Published:2023-07-15
  • Contact: Han Na, MD, Researcher, Department of Orthopedics and Trauma, Peking University People’s Hospital, Beijing 100044, China; Key Laboratory of Trauma and Neural Regeneration, Ministry of Education, Beijing 100044, China; National Center for Trauma Medicine, Beijing 101100, China
  • About author:Feng Ruiqin, Master candidate, Physician, Department of Orthopedics and Trauma, Peking University People’s Hospital, Beijing 100044, China; Key Laboratory of Trauma and Neural Regeneration, Ministry of Education, Beijing 100044, China; National Center for Trauma Medicine, Beijing 101100, China
  • Supported by:
    Natural Science Foundation of Beijing, No. 7222198 (to HN)

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摘要：


文题释义：

富血小板血浆：是通过离心或单采工艺从血液中富集血小板而获得，特征是拥有高血小板比例。富血小板血浆中的血小板激活后可以释放各种生长因子或细胞因子，这些因子对间充质干细胞的修复活动可能产生有利影响。另外近来有研究发现富血小板血浆中的血小板可以向间充质干细胞递送线粒体，改善间充质干细胞的促血管发生能力。
间充质干细胞：是一类分布广泛、多组织来源的细胞群体，具有自我更新和多向分化能力。研究显示，间充质干细胞能够分泌促进再生的物质，还能抑制过度的炎症反应，因此被尝试用于治疗组织损伤和某些炎症性疾病。目前研究人员已开展了多项利用间充质干细胞修复周围神经损伤的研究，并取得了一些进展。
细胞外囊泡：是由细胞释放的膜结合颗粒，携带有多种生物活性分子，包括脂质、蛋白质和各类RNA等。细胞外囊泡具有多种生物学功能，广泛参与细胞间通讯，在作为治疗性分子载体方面具有巨大潜力。有学者认为细胞外囊泡是间充质干细胞发挥作用的主要介质。


背景：富血小板血浆已被证明能够增强间充质干细胞活性并具有促血管再生能力。细胞外囊泡是间充质干细胞发挥作用的关键介质之一，但目前还不清楚富血小板血浆是否能影响细胞外囊泡的功能。

目的：探索富血小板血浆对骨髓间充质干细胞分泌的细胞外囊泡功能的影响，验证是否能够将富血小板血浆用作一种刺激剂来改善骨髓间充质干细胞修复周围神经损伤的效果。
方法：体外实验方面，在一般条件下和含有1%富血小板血浆的条件下培养骨髓间充质干细胞，用超速离心法分离骨髓间充质干细胞分泌的细胞外囊泡，前者命名为EVs-nor，后者命名为EVs-prp。将细胞外囊泡用于干预施万细胞，通过EdU、蛋白印迹、qPCR、光学显微镜拍照等分析施万细胞修复性重编程相关特征，包括细胞增殖、c-Jun蛋白表达、重编程相关基因表达、细胞形态等。体内实验方面，构建大鼠坐骨神经断伤模型，然后利用甲壳素神经套管将骨髓间充质干细胞单独或连同1%富血小板血浆植入损伤神经，术后8周评估组织学和功能学恢复情况，评估指标包括再生神经纤维密度、腓肠肌肌肉湿质量、坐骨神经功能指数等。

结果与结论：①相比于EVs-nor，EVs-prp刺激后的施万细胞增殖能力更强，c-Jun基因表达水平更高，GDNF表达上调，细胞形态更长，表明EVs-prp比EVs-nor有更强的刺激施万细胞重编程的能力；②坐骨神经损伤动物实验结果显示，相比于单独植入骨髓间充质干细胞或单独植入富血小板血浆，将骨髓间充质干细胞联合富血小板血浆一起植入的修复效果最好，具体体现在该组的再生神经纤维密度、腓肠肌肌肉湿质量、坐骨神经功能指数等指标均显著优于其他组；③这些实验结果显示，富血小板血浆可以改善骨髓间充质干细胞来源细胞外囊泡的功能，并且可以作为一种实用、易得的制剂与骨髓间充质干细胞协同促进周围神经损伤修复。

https://orcid.org/0000-0001-5995-5184(冯睿钦)；https://orcid.org/0000-0003-4590-3361(韩娜) 

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

关键词: 富血小板血浆, 间充质干细胞, 细胞外囊泡, 周围神经损伤, 施万细胞, 重编程, 神经导管, 间充质干细胞移植, 坐骨神经损伤

Abstract: BACKGROUND: Platelet-rich plasma has been shown to enhance the viability and the pro-angiogenesis capacity of mesenchymal stem cells. Extracellular vesicles are one of the key mediators for mesenchymal stem cells to exert their effects, but currently, it is unclear whether platelet-rich plasma affects the functions of extracellular vesicles.
OBJECTIVE: To investigate the effects of platelet-rich plasma on the function of extracellular vesicles from bone marrow mesenchymal stem cells, verify whether platelet-rich plasma can be used as an adjuvant to enhance the healing effects of bone marrow mesenchymal stem cells on repairing the peripheral nerve injury.
METHODS: For in vitro study, bone marrow mesenchymal stem cells were cultured under normal conditions and with 1% platelet-rich plasma. The ultracentrifugation was used to extract the extracellular vesicles produced by bone marrow mesenchymal stem cells cultured under normal conditions (EVs-nor) or the condition supplemented with 1% platelet-rich plasma (EVs-prp). Extracellular vesicles were used to incubate with Schwann cells. The EdU assay, western blot assay, qPCR and light microscopy photography were performed to examine the effects of EVs-nor and EVs-prp on Schwann cell reprogramming, which was characterized by cell proliferation, c-Jun expression, reprogramming-associated gene expression and cell morphology. For in vivo study, the model of sciatic nerve injury in rats was established. Bone marrow mesenchymal stem cells were grafted with or without 1% platelet-rich plasma into the injured rat sciatic nerve using a chitin nerve conduit. Eight weeks after the surgery, the recovery was assessed by histological and functional indexes, including regenerated nerve fiber density, gastrocnemius wet weight ratio and sciatic function index.
RESULTS AND CONCLUSION: (1) Compared with EVs-nor, EVs-prp was stronger in promoting Schwann cell proliferation. The gene expressions of c-Jun and GDNF were significantly upregulated in EVs-prp treated Schwann cells. The morphology of Schwann cells was significantly longer in EVs-prp group than that in EVs-nor group, indicating that EVs-prp had a stronger ability to stimulate Schwann cell reprogramming than EVs-nor. (2) Sciatic nerve injury animal experiment results revealed that grafting mesenchymal stem cells along with platelet-rich plasma into the injured sciatic nerve showed the best recovery compared with grafting mesenchymal stem cells or platelet-rich plasma alone, demonstrated by the significantly improved density of nerve fibers, gastrocnemius wet weight ratio, and sciatic function index. (3) These results suggested that platelet-rich plasma improved the function of bone marrow mesenchymal stem cell-derived extracellular vesicles and could be served as a practical and feasible preparation to synergize with bone marrow mesenchymal stem cells to improve peripheral nerve repair.

Key words: platelet-rich plasma, mesenchymal stem cell, extracellular vesicle, peripheral nerve injury, Schwann cell, reprogramming, nerve guide conduit, mesenchymal stem cell transplantation, sciatic nerve injury

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