摘要
[目的]探讨负载miRNA-27b的间充质干细胞来源的外泌体(MSC-27b-Exos)对炎症环境下的软骨细胞的保护作用。[方法]体外利用10 ng/ml白介素-1β(IL-1β)建立软骨细胞炎症环境模型,采用超速离心法收集外泌体,分别以80μg/ml的MSC-27b-Exos,普通外泌体MSC-Exos、inhibitor沉默的MSC-27boff-Exos作用炎症环境下的软骨细胞,并设PBS处理为对照组,检测各处理组软骨细胞凋亡水平,Western blot检测软骨细胞中MMP-13,及凋亡蛋白Caspase的表达水平。体内实验:20只雄性SD大鼠随机分为4组,建立创伤性骨关节炎(post-traumatic osteoarthritis, PTOA)模型,分别注射等量的MSC-miR27b-Exos、MSC-miR27b^(off)-Exos,并设立单纯PBS组和假手术组,6周后取材,观察关节软骨形态;ELISA检测关节液中IL-1β、TNF-α的含量;免疫组化对比各组Ⅱ型胶原表达。[结果]体外实验中,MSC-27b-Exos组的软骨细胞增殖能力较MSC-miR27b^(off)-Exos组增强,较空白组明显旺盛(P<0.05),在炎症环境下软骨细胞凋亡明显减弱(P<0.05),MSC-27b-Exos组软骨细胞中cleave-Caspase-9水平降低(P<0.05),cleave-Caspase-3水平降低(P<0.05),MMP-13相对含量减少。SD大鼠体内实验显示, MSC-27bExos组对软骨缺损修复效果优于空白组和MSC-miR27b^(off)-Exos组。MSC-27b-Exos组关节液中IL-1β、TNF-α的含量低于空白组,明显低于MSC-27b^(off)-Exos组。[结论]在实验型SD大鼠PTOA模型中,利用MSC-27b-Exos关节腔内注射具有明显抗炎的作用和减缓关节软骨退行性变。
[Objective]To explore the protective effect of mesenchymal stem cell-derived exosomes(MSC-27b-Exos)loaded with miRNA-27b on chondrocytes in inflammatory environment.[methods]The chondrocyte inflammatory environment model was established in vitro by using 10 ng/ml IL-1β.The exosomes were collected by ultracentrifugation respectively.After that,80μg/ml of MSC-27b-Exos,common exosomes MSC-Exos,inhibitor silenced MSC-27boff-Exos were acted on chondrocytes in inflammatory environment,and PBS was used as a control group to detect the apoptosis level of chondrocytes in each treatment group.Western blot was used to detect MMP-13 in chondrocytes,and the expression level of the apoptotic protein Caspase.In vivo experiment,20 male Sprague-Dawley rats were randomly divided into 4 groups to establish a model of traumatic osteoarthritis(PTOA),which were injected equal amounts of MSC-miR27b-Exos,MSC-miR27boffExos and PBS,additionally received sham operation.After 6 weeks,the articular cartilage morphology was observed.The levels of IL-1βand TNF-αin the joint fluid were detected by ELISA.Immunohistochemical assessment of the expression of type II collagen in each group was conducted.[Results]In vitro,the exogenous chondrocyte proliferation ability of MSC-27 bExos was significantly stronger than that of MSC-miR27 boff-Exos group and the blank group(P<0.05).Chondrocyte apoptosis was significantly attenuated in inflammatory environment(P<0.05),and caspase-9 level was decreased in MSC-27 b-Exos group(P<0.05),cleave-Caspase-3 level also decreased(P<0.05),as well as the relative content of MMP-13 decreased.In vivo,MSC-27 b-Exos group had better repair effect on cartilage defects than blank group and MSC-miR27 boff-Exos group.MSC-27 b-Exos group had significantly lower IL-1βand TNF-αin the joint fluid than the blank group,and MSC-27 boff-Exos group.[Conclusion]In the experimental SD rat PTOA model,intra-articular injection of Exos joint with MSC-27 b-Exos has obvious anti-inflammatory effect and alleviate articular cartilage degeneration.
作者
苏永蔚
周山健
肖大伟
田大川
罗华
刘丹平
綦慧
SU Yong-wei;ZHOU Shan-jian;XIAO Da-wei;TIAN Da-chuan;LUO Hua;LIU Dan-ping;QI Hui(Department of Orthopedics,The First Affiliated Hospital,Jinzhou Medical University,Jinzhou121000,China;Beijing Institute of Traumatology and Orthopedics,Beijing100035,China;Fengxian Hospital,Graduate Base,Jinzhou Medical University,Shanghai201499,China;The Second Department ofOrthopaedics,Fuyang Tumor Hospital,Fuyang,236000,China;Department of Orthopedics,People's Hospital of Jurong,Jurong212400,China)
出处
《中国矫形外科杂志》
CAS
CSCD
北大核心
2019年第8期726-734,共9页
Orthopedic Journal of China
基金
国家自然科学基金资助课题(编号:81572140)
北京市自然科学基金面上项目资助课题(编号:7172036)
作者简介
苏永蔚,在读硕士研究生,研究方向:关节软骨缺损修复,(电话)18531355324,(电子信箱)suyongwei0826@163.com;通信作者:刘丹平,(电话)0416-4197673,(电子信箱)liudanping2009@sohu.com;通信作者:綦惠,(电话)13810027092,(电子信箱)kellyqhqh2002@163.com
