摘要
目的探讨SD鼠骨髓间充质干细胞(BMSCs)来源外泌体(Exos)修复骺板缺损的相关机制。方法通过超离心提取鼠BMSCs来源的Exos,制备聚乳酸-羟基乙酸共聚物(PLGA)负载的Exos(PLGA-Exos)支架,扫描电镜对支架进行表征。提取鼠软骨细胞,探究PLGA-Exos/RAW264.7细胞/软骨细胞共培养体系中巨噬细胞及软骨细胞相关基因的表达。将24只SD大鼠随机均分为A、B、C 3组,构建胫骨近端骺板缺损模型,将PLGA负载的BMSCs-Exos缓释棒(A组)及无Exos负载的PLGA棒(B组)分别填塞于骺板缺损区,C组为对照组(无填塞)。术后6周获取鼠胫骨标本,通过影像学X线分析、苏木精-伊红(HE)染色、甲苯胺蓝染色、Ⅱ型胶原免疫组织化学染色及巨噬细胞表型蛋白[M1型:诱导型一氧化氮合酶(iNOS);M2型:Arg-1]染色,评估骺板缺损的修复效果。两组间比较采用独立样本t检验。结果PLGAs支架为三维多孔结构,孔隙分布均匀,具有较高的连通性。Exos被成功加载至PLGA中。PCR结果发现,在炎症微环境中,与对照组比较,BMSCs-Exos调控巨噬细胞高表达M2表型蛋白Arg-1(7.12±1.01比1.00±0.03,t=10.47,P<0.01),且促进软骨细胞COL-2及Sox9基因mRNA表达(8.25±0.74比1.00±0.12,t=16.84,P<0.01;4.64±0.16比1.00±0.01,t=38.64,P<0.01)。术后6周胫骨标本影像学及组织学分析显示,A组骺板缺损区可见结构致密、与正常骺板结构及功能相似新生透明软骨组织,B组骺板缺损区新生组织由大量的纤维组织及少量的透明软骨组成,C组骺板缺损区骨桥形成。骺板损伤区巨噬细胞相关的表型蛋白免疫组织化学染色显示,A组M2型巨噬细胞表型蛋白Arg-1染色呈阳性显色反应,M1型巨噬细胞表型蛋白iNOS染色为阴性;B组少量细胞质基质被Arg-1抗体染为弱阳性,iNOS染色为阴性;C组Arg-1抗体染色为阴性,而iNOS染色呈阳性反应。结论在炎症微环境中,BMSCs-Exos调控巨噬细胞向M2型编程,促进骺板缺损修复。
Objective To investigate the mechanism of exosomes(Exos)derived from bone marrow mesenchymal stem cells(BMSCs)repairing epiphyseal plate defects in SD rats.Methods Exos derived from young rat BMSCs was extracted by ultra-centrifugation,and the polylactic-co-glycolic acid(PLGA)-Exos scaffolds were prepared and characterized by scanning electron microscopy.The chondrocytes of SD rats were extracted to explore the expression of macrophage and chondrocyte related genes in PLGA-ExOS/RAW264.7 cells/chondrocytes co-culture system.A total of 24 SD rats were randomly divided into groups A,B,C,and the proximal tibial epiphyseal plate defect model was constructed.PLGA-Exos sustained release rods(group A)and PLGA rods without Exos load(group B)were stuffed into the epiphyseal plate defect area,and group C was the control group without treatment.At 6th week after operation,tibia specimens were collected and analyzed by X-ray imaging,hematoxylin-eosin(HE)staining,toluidine blue staining,typeⅡcollagen immunohistochemical staining and macrophage phenotype protein[M1 macrophages:inducible nitric oxide synthase(iNOS);M2 macrophages:ARG-1]staining were used to evaluate the repair effect of epiphyseal plate defects.Results are presented as mean±standard deviation.Statistical analysis was performed using independent-t test.Results The PLGA scaffold was a three-dimensional porous structure with uniform pore distribution and high connectivity.And Exos were successfully loaded into PLGA.PCR results showed that BMSCs-Exos regulated the expression of M2 phenotypic protein Arg-1 in macrophages(7.12±1.01 vs.1.00±0.03,t=10.47,P<0.01),and promoted the mRNA expression of COL-2 and Sox9 genes in chondrocytes(8.25±0.74 vs.1.00±0.12,t=16.84,P<0.01;4.64±0.16 vs.1.00±0.01,t=38.64,P<0.01).At 6th week after surgery,the imaging and histological analysis of tibial specimens indicated that the structure and function of new hyaline cartilage tissue in the epiphyseal plate defect area were similar to the normal epiphyseal plate in group A;the new tissue in the epiphyseal plate defect area was composed of a large amount of fibrous tissue and a small amount of hyaline cartilage in group B;and the bone bridge was formed in the epiphyseal plate defect area in group C.Immunohistochemical staining of macrophage-related phenotypic proteins in the epiphyseal plate injury area suggested that Arg-1 staining of M2 macrophages biomarker in group A was positive and iNOS staining of M1 macrophages biomarker was negative.In group B,a small amount of cytoplasmic matrix was weakly positive by Arg-1 antibody,but negative by iNOS.In group C,Arg-1 antibody staining was negative,but iNOS staining was positive.Conclusion In inflammatory microenvironment,BMSCs-Exos regulate macrophage programming to M2 type and promote the repair of epiphyseal plate defects.
作者
邓玲珑
成昊
余黎
朱纯权
陶圣祥
邓凯
Deng Linglong;Cheng Hao;Yu Li;Zhu Chunquan;Tao Shengxiang;Deng Kai(Department of Orthopaedic Trauma and Microsurgery,Zhongnan Hospital of Wuhan University,Wuhan 430071,China;National Demonstration Center for Experimental General Medicine Education Xianning Medical College,Hubei University of Science and Technology,Xianning 437000,China)
出处
《中华实验外科杂志》
CAS
北大核心
2022年第9期1721-1724,共4页
Chinese Journal of Experimental Surgery
关键词
骨髓间充质干细胞
外泌体
巨噬细胞
骺板
修复
Bone marrow mesenchymal stromal cells
Exosome
Macrophages
Epiphyseal plate
Repair