二甲双胍修饰骨髓间充质干细胞外泌体调节软骨细胞的作用及机制  

Effect and mechanism of metformin-modified bone marrow mesenchymal stem cell exosomes on regulating chondrocytes

作　　者：司马鑫利 刘丹平 綦惠[2] Sima Xinli;Liu Danping;Qi Hui(First Affiliated Hospital,Jinzhou Medical University,Jinzhou 121000,Liaoning Province,China;Beijing Trauma Orthopedics Institute,Beijing Jishuitan Hospital,Beijing 100035,China)

机构地区：[1]锦州医科大学附属第一医院,辽宁省锦州市121000 [2]北京市创伤骨科研究所,北京积水潭医院,北京市100035

出　　处：《中国组织工程研究》2025年第36期7728-7734,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金项目(81572140),项目负责人:刘丹平;北京市自然科学基金面上项目(7172036),项目负责人:綦惠。

摘　　要：背景:骨关节炎是一种以软骨基质持续性破坏为特征的退行性关节病,目前仍无有效的药物治疗方案,二甲双胍预处理骨髓间充质干细胞来源外泌体因避免了口服药物不良反应及免疫原性等特点,可能成为治疗骨关节炎的新方法。目的:探讨二甲双胍预处理骨髓间充质干细胞来源外泌体对软骨细胞的调节作用。方法:体外培养乳兔骨髓间充质干细胞和软骨细胞。使用超速离心法收集正常骨髓间充质干细胞源性外泌体和经过二甲双胍预处理的骨髓间充质干细胞源性外泌体,分别用含外泌体的培养基培养软骨细胞24 h,再用100μmol/LH_(2)O_(2)干预24 h。采用CCK-8法和划痕愈合实验检测软骨细胞增殖和迁移能力变化;采用Western blot和RT-qPCR检测软骨细胞中Ⅱ型胶原、P16蛋白及mRNA的表达变化,以及采用Western blot检测MKK7/JNK通路蛋白的表达变化;使用ELISA试剂盒检测软骨细胞中超氧化物歧化酶活性和丙二醛水平。结果与结论:(1)在氧化应激环境下,软骨细胞的增殖和迁移能力减弱,两种外泌体能够一定程度恢复软骨细胞的增殖和迁移能力,二甲双胍预处理骨髓间充质干细胞来源外泌体的改善效果更显著(P<0.05);(2)与正常骨髓间充质干细胞来源外泌体相比,二甲双胍预处理骨髓间充质干细胞来源外泌体更能有效提高Ⅱ型胶原表达和超氧化物歧化酶活性(P <0.05),同时也更能有效降低P16表达和丙二醛水平(P <0.05);(3)两种外泌体能够一定程度抑制MKK7和p-JNK蛋白表达,二甲双胍预处理骨髓间充质干细胞来源外泌体的抑制效果更显著(P<0.05)。结果表明,在氧化应激环境下,二甲双胍预处理骨髓间充质干细胞来源外泌体通过抑制MKK7/JNK通路对抗软骨细胞衰老并促进软骨细胞增殖。BACKGROUND:Osteoarthritis is a progressive joint condition identified by ongoing deterioration of the cartilage matrix,and there is currently no effective drug treatment plan.Metformin-modified exosomes isolated from bone marrow-derived mesenchymal stem cells can become a new method for treating osteoarthritis due to their avoidance of oral drug adverse reactions and immunogenicity.OBJECTIVE:To study the controlling impact of exosomes from metformin-altered bone marrow-derived mesenchymal stem cells on chondrocytes.METHODS:Rabbit bone marrow-derived mesenchymal stem cells and chondrocytes were cultured in vitro.Bone marrow-derived mesenchymal stem cells derived exosomes and metformin pretreated bone marrow-derived mesenchymal stem cells derived exosomes were collected using a high-speed centrifuge.Chondrocytes were cultured with exosome-containing culture medium for 24 hours and then treated with 100μmol/L H_(2)O_(2) for 24 hours.The capability changes of two extracellular vesicles on chondrocyte proliferation and migration were detected using CCK8 assay and scratch healing experiment,respectively.Western blot analysis and RT-qPCR were employed to examine the alterations in the expression of type II collagen,P16 protein,and their mRNA in chondrocytes.Western blot analysis was utilized to assess the changes in the expression of MKK7/JNK pathway proteins.ELISA kits were utilized to measure the activity of cell superoxide dismutase and the levels of malondialdehyde in chondrocytes.RESULTS AND CONCLUSION:(1)In an oxidative stress environment,the proliferation and migration abilities of chondrocytes were weakened.The two types of exosomes could restore the proliferation and migration abilities of chondrocytes to a certain extent.Metformin pretreated bone marrow-derived mesenchymal stem cells derived exosomes had a significantly better improvement effect(P<0.05).(2)Compared with normal bone marrow mesenchymal stem cell-derived exosomes,metformin pretreated bone marrow-derived mesenchymal stem cells derived exosomes co

关 键 词：骨髓间充质干细胞 二甲双胍 外泌体 软骨细胞 骨关节炎 氧化应激 衰老 工程化干细胞 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学] R681.3