间充质干细胞来源的外泌体对肌腱细胞损伤修复的影响及其机制  

Effect and mechanism of mesenchymal stem cell derived exosomes in repair of tendon cell injury

作　　者：赵海波 赵夏[1] 高甲科[1] 宋文联 单正宜 于腾波[1] 张英泽[3] Zhao Haibo;Zhao Xia;Gao Jiake;Song Wenlian;Shan Zhengyi;Yu Tengbo;Zhang Yingze(Department of Sports Medicine,Affiliated Hospital of Qingdao University,Qingdao 266000,China;Department of Pathology,The Affiliated Hospital of Qingdao University,Qingdao 266000,China;Trauma Emergency Center,Third Hospital of Hebei Medical University,Shijiazhuang 050051,China)

机构地区：[1]青岛大学附属医院运动医学科,266000 [2]青岛大学附属医院病理科,266000 [3]河北医科大学第三医院创伤急救中心,石家庄050051

出　　处：《中华创伤杂志》2021年第7期653-661,共9页Chinese Journal of Trauma

基　　金：山东省自然科学基金(ZR2019MH097)

摘　　要：目的探讨人脐带间充质干细胞(hUC-MSCs)分泌的外泌体对损伤后肌腱细胞修复的影响及其机制。方法组织块贴壁法分离并纯化筛选出能稳定传代培养的hUC-MSCs,倒置荧光显微镜观察细胞形态,通过流式细胞术检测hUC-MSCs的免疫表型。应用诱导培养基诱导hUC-MSCs向成骨细胞、成软骨细胞和脂肪细胞分化并进行鉴定。运用高速离心法提取MSCs的分泌物外泌体(MSCs外泌体),用Western blot技术和电镜检测外泌体,用PKH67染色荧光法检测外泌体膜融合能力。40只Wistar鼠按随机数字表法分为肌腱损伤组和正常组,每组20只。肌腱损伤组:切断跟腱1周后用100 mg/kg戊巴比妥钠处死,取跟腱组织用胰蛋白酶消化得到损伤肌腱细胞。正常组:不做任何处理直接处死,与损伤组并行处理得到正常肌腱细胞。将外泌体与体外培养的肌腱细胞共培养,12,24,48,72 h后通过细胞计数CCK-8检测肌腱细胞增殖情况。hUC-MSCs外泌体处理细胞24 h后,采用Western blot、qPCR、免疫荧光方法检测外泌体对肌腱细胞转化生长因子-β(TGF-β)、骨形态发生蛋白(BMP)、血管内皮生长因子(VEGF)、成纤维细胞生长因子(FGF)和炎性因子白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)的影响。结果鉴定了hUC-MSCs,并成功分离hUC-MSCs外泌体。分离培养的MSCs呈梭形,丙氨酸氨肽酶(CD13)、整联蛋白β-1(CD29)、ecto-5’-核苷酸酶(CD73)、胸腺细胞表面抗原(CD90)和内皮素(CD105)呈阳性,人类白细胞DR抗原(HLA-DR)、造血祖细胞抗原(CD34)、白细胞共同抗原(CD45)呈阴性。分离出的外泌体经PKH67染色鉴定证实,在电镜下呈直径30~100 nm的圆盘、内部凹陷结构,Western blot检测高表达移动相关蛋白-1(CD9)和溶酶体相关膜蛋白3(CD63)。处理肌腱细胞后,CCK-8检测12,24,48,72 h细胞活性,结果表明肌腱损伤组显著高于正常组(P < 0.01),qPCR结果表明肌腱损伤组TGF-β(1.850 ± 0.127)、BMP(2.133 ± 0.Objective To investigate the effects and mechanism of exosomes secreted by human umbilical cord-derived mesenchymal stem cells(hUC-MSCs)in repair of tendon cell injury.Methods The hUC-MSCs which were stably subcultured were isolated and purified by a tissue block adherent method,and the immunophenotype of hUC-MSCs was detected by flow cytometry.The induction media was employed to induce the differentiation of hUC-MSCs to osteoblasts,chondroblasts and adipocytes,and cell identification was performed subsequently.The secreted exosomes of MSCs(MSCs-exosomes)were extracted using an ultracentrifugation method.The exosomes were detected by Western blot and electron microscopy,and the fusion ability of the exosome membrane was detected by PKH67 staining fluorescence.Forty Wistar rats were divided into tendon injury group(n=20)and normal group(n=20)according to the random number table.In tendon injury group,the rats were sacrificed with 100 mg/kg pentobarbital sodium one week after Achilles tendon transection,and the injured tendon cells were obtained following digestion of the Achilles tendon.In normal group,the rats were sacrificed without any treatment and the normal tendon cells were obtained concurrently.After the exosomes were co-cultured with tendon cells in vitro for 12,24,48,72 hours,the proliferation of tendon cells was detected by CCK-8 assay.After the tendon cells were treated with hUC-MSCs exosomes for 24 hours,the effects of exosomes on transforming growth factor B(TGF-0),bone morphogenetic protein(BMP),vascular endothelial growth factor(VEGF),fibroblast growth factor(FGF),interleukin(IL)-1βand tumor necrosis factor-α(TNF-α)were detected by Western blot,qPCR and immunofluorescence.Results The hUC-MSCs were identified and hUC-MSCs-exosomes were isolated successfully.The cultured MSCs were fusiform and positive for Alanine aminopeptidase(CD13),integrin 0-1(CD29),ECTO-5'-nucleotidase(CD73),thymocyte surface antigen(CD90)and endothelin(CD105),but negative for human leukocyte DR antigen(HLA-DR),hematopoietic

关 键 词：间质干细胞 外泌体 细胞因子类 肌腱细胞 

分 类 号：R686.1[医药卫生—骨科学]