摘要
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。
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.
作者
冯睿钦
韩娜
张蒙
谷馨怡
张丰识
Feng Ruiqin;Han Na;Zhang Meng;Gu Xinyi;Zhang Fengshi(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)
出处
《中国组织工程研究》
CAS
北大核心
2024年第7期985-992,共8页
Chinese Journal of Tissue Engineering Research
基金
北京市自然科学基金(7222198),项目负责人:韩娜。
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
platelet-rich plasma
mesenchymal stem cell
extracellular vesicle
peripheral nerve injury
Schwann cell
reprogramming
nerve guide conduit
mesenchymal stem cell transplantation
sciatic nerve injury
