锶离子外泌体促间充质干细胞成骨分化和内皮细胞血管生成的实验研究  

The study of strontium-induced MSC-derived exosomes in promoting osteogenic differentiation of mesenchymal stem cells and angiogenesis of endothelial cells

作　　者：杨思敏 殷实 周名亮 江飞[3] 蒋欣泉[1,2] YANG Simin;YIN Shi;ZHOU Mingliang;JIANG Fei;JIANG Xinquan(Department of Prosthodontics,Shanghai Ninth People's Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200011,China;National Clinical Research Center of Stomatology,National Clinical Research Center for Oral Diseases,Shanghai Key Laboratory of Stomatology,Shanghai Research Institute of Stomatology,Shanghai 200011,China;Department of General Dentistry,The Affiliated Stomatological Hospital of Nanjing Medical University,Nanjing 210029,China)

机构地区：[1]上海交通大学医学院附属第九人民医院口腔修复科,上海交通大学口腔医学院,上海200011 [2]国家口腔医学中心,国家口腔疾病临床医学研究中心,上海市口腔医学重点实验室,上海市口腔医学研究所,上海200011 [3]南京医科大学附属口腔医院综合诊疗科,江苏南京210029

出　　处：《口腔生物医学》2024年第2期66-74,共9页Oral Biomedicine

基　　金：国家自然科学基金创新研究群体(81921002);国家自然科学基金重点项目(82130027);国家自然科学基金青年项目(31900971)。

摘　　要：目的:探究锶离子诱导间充质干细胞(MSC)分泌的外泌体(以下简称为锶离子外泌体)对成骨分化和血管生成功能的影响。方法:向骨髓间充质干细胞(BMSC)中加入锶离子并通过超速离心法提取锶离子外泌体,以未经处理的BMSC外泌体作为对照。通过外泌体荧光标记观察其细胞内化作用;采用碱性磷酸酶(ALP)染色、ALP活性测定、实时荧光定量PCR等实验探究分离的外泌体对BMSC成骨分化的影响;采用细胞划痕实验、迁移实验、内皮细胞成管实验等方法评价外泌体对人脐静脉内皮细胞(HUVEC)体外血管形成能力的调控作用;利用miRNA测序探讨锶离子外泌体促进成骨和成血管作用的潜在分子机制。结果:锶离子刺激不会影响BMSC分泌外泌体,且分泌的锶离子外泌体可以被BMSC摄取内化,与对照组外泌体相比,锶离子外泌体可以增强BMSC的ALP活性(P<0.05),上调其ALP和Runt相关转录因子2(Runx2)的转录(P<0.05);同时锶离子外泌体可以促进HUVEC细胞迁移和小管形成(P<0.05),并提高血管内皮生长因子(VEGF)、促血管生成素1(ANG1)和成纤维细胞生长因子(FGF)的转录(P<0.05);外泌体miRNA测序发现锶离子刺激会引起BMSC外泌体内miRNA的差异性表达,差异表达的miR-125b-5p、miR-132-3p、miR-31a-5p以及miR-450b可能参与锶离子外泌体促成骨和成血管的功能发挥。结论:锶离子诱导MSC分泌的外泌体具有促进成骨分化和血管生成的双重功能。Objective:To explore the effect of mesenchymal stem cell(MSC)derived exosomes induced by strontium on osteogenic differentiation and angiogenesis,providing a new idea for the repair of vascularized bone defects.Methods:Strontium was added to bone marrow mesenchymal stem cell(BMSC)and strontium-exosomes were then extracted by ultracentrifugation,using untreated BMSC exosomes as control.The cell internalization was observed by fluorescent labeling of exosomes.Alkaline phosphatase(ALP)staining,ALP activity determination and qRT-PCR analysis were used to study the effects of exosomes on the osteogenic differentiation of BMSC.The effects of exosomes on vascular formation of endothelial cells in vitro were evaluated by wound healing assay,Transwell assay,tube formation assay and other methods.Finally,the potential mechanism of strontium-exosomes in promoting osteogenesis and angiogenesis was discussed by the miRNA sequencing.Results:The stimulation of strontium ion could not affect the secretion of exosomes by BMSC,and the strontium-exosomes could be internalized by BMSC.Strontium-exosomes could enhance the ALP activity of BMSC(P<0.05)and up-regulate the transcription of osteogenesis-related genes ALP and runt-related transcription factor 2(Runx2)(P<0.05).Strontium-exosomes could also promote the migration and the tubule formation of endothelial cells,and significantly increase the transcription of angiogenic-related genes of VEGF,ANG1 and FGF(P<0.05).The miRNA sequencing analysis revealed that the stimulation of strontium ions induced the differential expression of miRNA in exosomes derived from BMSC.The differential expression of miR-125b-5p,miR-132-3p,miR-31a-5p and miR-450b may be then involved in promoting osteogenic differentiation and angiogenesis of cells.Conclusions:The strontium-exosomes have dual functions of promoting angiogenesis and osteogenic differentiation.

关 键 词：骨组织工程 锶离子 外泌体 成骨分化 血管生成 

分 类 号：R780.2[医药卫生—口腔医学]