负载miRNA-27b的BMSCs来源的外泌体治疗实验性骨关节炎
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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, 80 μg/ml of MSC-27b-Exos, common exosomes MSC-Exos, inhibitor silenced MSC-27b~(off)-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-miR27b~(off)Exos and PBS, additionally received sham operation. After 6 weeks, the articular cartilage morphology was observed. The levels of IL-1β and TNF-α in 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-1β and TNF-α in 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.
[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, 80 μg/ml of MSC-27b-Exos, common exosomes MSC-Exos, inhibitor silenced MSC-27b~(off)-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-miR27b~(off)Exos and PBS, additionally received sham operation. After 6 weeks, the articular cartilage morphology was observed. The levels of IL-1β and TNF-α in 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-1β and TNF-α in 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.
引文
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[16]Coleman MC,Goetz JE,Brouillette MJ,et al.Targeting mitochondrial responses to intra-articular fracture to prevent posttraumatic osteoarthritis[J].Sci Transl Med,2018,10(427):5372.
[17]Mabey T,Honsawek S.Cytokines as biochemical markers for knee osteoarthritis[J].World J Orthop,2015,6(1):95-105.
[18]Wang X,Hunter D,Xu J,et al.Metabolic triggered inflammation in osteoarthritis[J].Osteoarthritis,2015,23(1):22-30.
[19]Kapoor M,Martel-Pelletier J,Lajeunesse D,et al.Role of proinflammatory cytokines in the pathophysiology of osteoarthritis[J].Nat Rev Rheumatol,2011,7(1):33-42.
[20]Lewis JJ,Furman BD,Zeitler E,et al.Genetic and Cellular Evidence of Decreased Inflammation Associated with Reduced Incidence of Posttraumatic Arthritis in MRL/MpJ Mice[J].Arthritis Rheum,2013,3:660-670.
[21]Glynn SA,Klein HG,Ness PM.The red blood cell storage lesion:the end of the beginning[J].Transfusion,2016,56(6):1462-1468.
[22]Zhang YL,Min soo Kim,Jia BS,et al.Hypothalamic stem cells control ageing speed partly through exosomal miRNAs[J].Nature,2017,548(7665):52.
[23]Rani S,Ryan AE,Griffin MD,et al.Mesenchymal stem cell-derived extracellular vesicles:toward cell-free therapeutic applications[J].Molecular Ther,2015,23(5):812.
[24]綦惠,靳少锋,陈磊,等.富血小板血浆复合再生支架修复兔骨软骨缺损[J].中国矫形外科杂志,2018,26(8):740-745.
[2]Cheleschi S,De A P,Pecorelli A,et al.Hydrostatic pressure regulates microRNA expression levels in osteoarthritic chondrocyte cultures via the wnt/β-catenin pathway[J].Int J Molec Sci,2017,18(1):133
[3]Sieghart D,Liszt M,Wanivenhaus A,et al.Hydrogen sulphide decreases IL-1β-induced activation of fibroblast-like synoviocytes from patients with osteoarthritis[J].J Cellular Molecular Med,2015,19(1):187.
[4]Mabey T,Honsawek S.Cytokines as biochemical markers for knee osteoarthritis[J].World J Orthop,2015,6(1):95-105.
[5]Wang X,Hunter D,Xu J,et al.Metabolic triggered inflammation in osteoarthritis[J].Osteoarthritis,2015,23(1):22-30.
[6]Kapoor M,Martel-Pelletier J,Lajeunesse D,et al.Role of proinflammatory cytokines in the pathophysiology of osteoarthritis[J].Nat Rev Rheumatol,2011,7(1):33-42.
[7]Zhen GH,Wen CY,Jia XF,et al.Inhibition of TGF-βsignaling in subchondral bone mesenchymal stem cells attenuates osteoarthritis[J].Nature Med,2016,19(6):704-712.
[8]包德明,吕秉舒,李东升,许京华,吕秉乐.骨髓间充质干细胞浓缩上清液对骨性关节炎大鼠模型治疗作用的初步研究[J].中国矫形外科杂志,2015,23(21):1979-1984.
[9]Lai R C,Arslan F,Lee M M,et al.Exosome secreted by MSC reduces myocardial ischemia/reperfusion injury[J].Stem Cell Research,2010,4(3):214.
[10]Farh KK,Grimson A,Jan C,et al.The widespread impact of mammalian MicroRNAs on m RNA repression and evolution[J].Science,2005,310(5755):1817-1821.
[11]田大川,李海乐,肖大伟,等.联合应用软骨再生支架与突变型HIF-1α修饰BMSCs分泌的外泌体对晚期软骨缺损修复的促进作用[J].吉林大学学报(医学版),2018,44(02):216-222,461.
[12]Maes C,Araldi E,Haigh K,et al.VEGF independent cell autonomous functions of HIF 1αregulating oxygen consumption in fetal cartilage are critical for chondrocyte survival[J].Jo Bone Mineral Res,2012,27(3):596-609.
[13]Akhtar N,Rasheed Z,Ramamurthy S,et al.MicroRNA-27b regulates the expression of matrix metalloproteinase 13 in human osteoarthritis chondrocytes[J].Arthritis Rheumatism,2010,62(5):1361-1371.
[14]陈晓东,林建华.大鼠关节软骨细胞的分离、培养与鉴定[J].福建中医学院学报,2006(6):27-29,63.
[15]丁晓飞.大鼠骨髓间充质干细胞分离、体外培养和向软骨细胞表型定向分化的实验研究[C].2005'中国修复重建外科论坛论文汇编,2005:2.
[16]Coleman MC,Goetz JE,Brouillette MJ,et al.Targeting mitochondrial responses to intra-articular fracture to prevent posttraumatic osteoarthritis[J].Sci Transl Med,2018,10(427):5372.
[17]Mabey T,Honsawek S.Cytokines as biochemical markers for knee osteoarthritis[J].World J Orthop,2015,6(1):95-105.
[18]Wang X,Hunter D,Xu J,et al.Metabolic triggered inflammation in osteoarthritis[J].Osteoarthritis,2015,23(1):22-30.
[19]Kapoor M,Martel-Pelletier J,Lajeunesse D,et al.Role of proinflammatory cytokines in the pathophysiology of osteoarthritis[J].Nat Rev Rheumatol,2011,7(1):33-42.
[20]Lewis JJ,Furman BD,Zeitler E,et al.Genetic and Cellular Evidence of Decreased Inflammation Associated with Reduced Incidence of Posttraumatic Arthritis in MRL/MpJ Mice[J].Arthritis Rheum,2013,3:660-670.
[21]Glynn SA,Klein HG,Ness PM.The red blood cell storage lesion:the end of the beginning[J].Transfusion,2016,56(6):1462-1468.
[22]Zhang YL,Min soo Kim,Jia BS,et al.Hypothalamic stem cells control ageing speed partly through exosomal miRNAs[J].Nature,2017,548(7665):52.
[23]Rani S,Ryan AE,Griffin MD,et al.Mesenchymal stem cell-derived extracellular vesicles:toward cell-free therapeutic applications[J].Molecular Ther,2015,23(5):812.
[24]綦惠,靳少锋,陈磊,等.富血小板血浆复合再生支架修复兔骨软骨缺损[J].中国矫形外科杂志,2018,26(8):740-745.