低氧培养BMSCs的外泌体促进软骨再生的机制研究  

Exosomes secreted by hypoxia-preconditioned BMSCs promote cartilage regeneration

作　　者：沈凯 翟晨骏 左强[1] 梁文卫 范卫民[1] 刘锋[1] Shen Kai;Zhai Chenjun;Zuo Qiang;Liang Wenwei;Fan Weimin;Liu Feng(Department of Orthopaedics,First Affiliated Hospital of Nanjing Medical University,Nanjing 210029,China;Department of Orthopaedics,Affiliated Yixing Hospital of Jiangsu University,Yixing 214221,China)

机构地区：[1]南京医科大学第一附属医院骨科,南京210029 [2]江苏大学附属宜兴医院骨科,宜兴214221

出　　处：《中华骨科杂志》2023年第12期831-840,共10页Chinese Journal of Orthopaedics

基　　金：国家自然科学基金项目(81974332,82202716)。

摘　　要：目的探讨低氧培养骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)来源的外泌体中miR-196b-5p对软骨细胞功能的影响及作用机制,并分析其在软骨再生修复中的应用。方法将软骨细胞分别在常氧培养和低氧培养的BMSCs上清液中培养,通过CCK-8检测软骨细胞的增殖能力,qPCR检测Ⅱ型胶原(collagen type 2,Col2)、Col1、Aggrecan、SOX9的表达情况,评估低氧培养后BMSCs的外泌体对软骨细胞功能的影响。通过超速离心法获取常氧外泌体和低氧外泌体,采用CCK-8和qPCR检测对比低氧BMSCs外泌体和常氧BMSCs外泌体对软骨细胞增殖及合成代谢相关基因表达的促进作用。验证外泌体miRNA测序结果中低氧外泌体miR-196b-5p的表达情况,敲除低氧BMSCs中miR-196b-5p进行功能抑制实验,对比miR-196b-5p在低氧外泌体促进软骨细胞功能中发挥的作用。使用生物信息学工具预测并验证miR-196b-5p下游靶点。通过转染siRNA分析低氧BMSCs外泌体中miR-196b-5p促进软骨细胞功能的作用机制。采用丝素水凝胶负载低氧外泌体及低表达miR-196b-5p的低氧外泌体,注入裸鼠皮下后培养4周后对取出的组织块行番红O、马松组织学染色及硫酸糖胺聚糖、胶原生化含量检测,评估低氧外泌体及miR-196b-5p在软骨再生修复中的应用情况。结果低氧组软骨细胞吸光度为1.20±0.07,较常氧组的0.94±0.04高,差异有统计学意义(P<0.05);低氧组软骨细胞Col2(2.95±0.17)、Aggrecan(2.45±0.27)、SOX9(2.92±0.29)的表达高于常氧组(1.89±0.09、1.67±0.21、1.76±0.16),差异有统计学意义(P<0.05)。CCK-8示低氧外泌体组软骨细胞的增殖能力(1.28±0.04)较常氧外泌体组(1.05±0.06)更高,差异有统计学意义(P<0.05)。PmirGLO-BACH1-WT报告载体与miR-196b-5p mimics共转染后(0.73±0.06)的荧光素酶活性降低,差异有统计学意义(P<0.05);PmirGLO-BACH1-MUT报告载体与miR-196b-5p mimics共转染后,荧光素酶活性无变化,BACH1为Objective Observing the effect of exosomes derived from hypoxic Bone marrow mesenchymal stem cells(BMSCs)on the function of chondrocytes,and exploring the role and mechanism of exosomal miR-196b-5p.Evaluating the application prospects of hypoxic BMSCs exosomes and miR-196b-5p for cartilage regeneration.Methods Chondrocytes were cultured in the supernatant of BMSCs cultured under normoxia or hypoxia,respectively.The proliferation of chondrocytes was detected by CCK-8 assay and the expressions of Collagen type 2(Col2),Col1,Aggrecan and SOX9 were detected by qPCR to evaluate the effect of hypoxic BMSCs paracrine on chondrocyte functions.Obtaining normoxic and hypoxic exosomes through ultracentrifugation,and testing their effects on the proliferation and anabolic-related genes of chondrocytes through CCK-8 assay and qPCR.Verifying the expression of miR-196b-5p in hypoxic exosomes based on exosomal miRNA array.Knocking out miR-196b-5p in hypoxic BMSCs,and detecting the effect of hypoxic exosomal miR-196b-5p on the functions of chondrocytes by loss-of-function assay.Predicting the downstream of miR-196b-5p through bioinformatics tools,and exploring the mechanism of hypoxic exosomal miR-196b-5p by gain-of-function assays.Hypoxic exosomes and miR-196b-5p-knockout hypoxic exosomes were loaded on silk fibroin hydrogel and subcutaneously into nude mice.After 4 weeks of culture,histological staining of saffron O,Masson and biochemical content of sGAG and collagen were performed to assess the application prospect of hypoxic exosomes and hypoxic exosomal miR-196b-5p on cartilage regeneration.Results The results of CCK-8 assay and qPCR indicated that the supernatant of hypoxic BMSCs significantly promoted the proliferation of chondrocytes 1.20±0.07 and the expression of cartilage-related markers(Col22.95±0.17,Aggrecan 2.45±0.27,SOX92.92±0.29)compared to normoxic BMSCs(0.94±0.04,1.89±0.09,1.67±0.21,1.76±0.16),the differences were statistically significant(P<0.05).The result of CCK-8 assay showed that hypoxic exosomes(1.2

关 键 词：骨髓间充质干细胞 低氧 外泌体 miR-196b-5p 软骨再生 

分 类 号：R681[医药卫生—骨科学]