不同来源间充质干细胞外泌体治疗骨关节炎疗效的比较

Comparison of effects of exosomes secreted by different mesenchymal stem cells for the treatment of osteoarthritis

背景:在过去的数十年中,间充质干细胞作为组织工程种子细胞,在骨关节炎治疗中展现出了强大的疗效,在国内已开展了多项临床注册试验。研究表明,间充质干细胞主要依靠旁分泌效应释放外泌体等载体在体内发挥治疗作用。目的:比较骨髓间充质干细胞来源外泌体和脐带间充质干细胞来源外泌体治疗骨关节炎的效果。方法:采用超速离心法提取骨髓间充质干细胞来源外泌体和脐带间充质干细胞来源外泌体,采用透射电镜、Nanosight和Western blot鉴定两种外泌体;采用白细胞介素1β与软骨细胞共培养体外模拟骨关节炎环境,通过CCK-8、Western blot、qPCR评估两种外泌体对骨关节炎软骨细胞增殖、凋亡和细胞外基质分泌的影响;建立Ⅱ型胶原酶诱导的大鼠骨关节炎模型,通过大体和组织学染色评估体内注射两种外泌体治疗骨关节炎的疗效,ELISA检测关节液中白细胞介素1β和肿瘤坏死因子α水平。结果与结论:①两种外泌体在透射电镜下均为典型的圆形或杯状,粒径在105-120 nm之间,均表达CD63、CD81和TSG1010特征性蛋白;②脐带间充质干细胞来源外泌体的产量和纯度均高于骨髓间充质干细胞来源外泌体;③两种外泌体均能缓解白细胞介素1β对软骨细胞的炎症作用,主要表现在促进增殖、抑制凋亡和增加细胞外基质的分泌,其中脐带间充质干细胞来源外泌体的促软骨修复活性更强;④两种外泌体治疗后,骨关节炎大鼠关节软骨的宏观表现和OARSI评分均高于对照组,且关节液中炎症因子水平下降,其中脐带间充质干细胞来源外泌体的疗效更优;⑤结果表明:脐带间充质干细胞来源外泌体的产量和纯度均高于骨髓间充质干细胞来源外泌体,且脐带间充质干细胞来源外泌体对骨关节炎的治疗效果优于骨髓间充质干细胞来源外泌体,因此脐带间充质干细胞来源外泌体更适合作为治疗骨关节炎的新疗法。

BACKGROUND:Mesenchymal stem cells,as seed cells in tissue engineering,have shown powerful curative effect for the treatment of osteoarthritis in the past few decades,and many registered clinical experiments have been carried out in China.More studies have shown that mesenchymal stem cells that play a therapeutic role in vivo release exosomes through paracrine function.OBJECTIVE:To compare the effect of bone marrow mesenchymal stem cell exosomes and umbilical cord mesenchymal stem cell exosomes on the treatment of osteoarthritis.METHODS:Bone marrow mesenchymal stem cell exosomes and umbilical cord mesenchymal stem cell exosomes were isolated by ultra-centrifugation.Nanosight,transmission electron microscopy,and western blot assay were used to identify exosomes.Chondrocytes were co-cultured with interleukin-1βto simulate osteoarthritis in vitro.The effects of bone marrow mesenchymal stem cell exosomes and umbilical cord mesenchymal stem cell exosomes on the proliferation,apoptosis and extracellular matrix secretion of chondrocytes were evaluated by CCK-8 assay,western blot assay,and qPCR.Collagen II collagenase was injected to induce rat osteoarthritis model and the efficacy of in vivo injection of exosomes was evaluated by macroscopic evaluation and histological staining.The concentrations of interleukin-1βand tumor necrosis factor-αof joint fluid were examined by ELISA.RESULTS AND CONCLUSION:(1)Both kinds of exosomes were cup-shaped or round-shaped with particle size of 105-120 nm,and expressed characteristic proteins CD63,CD81,and TSG1010.(2)The yield and purity of umbilical cord mesenchymal stem cell exosomes were higher than those of bone marrow mesenchymal stem cell exosomes.(3)Both exosomes could alleviate the inflammatory effect of interleukin-1βon chondrocytes,which was mainly manifested in promoting proliferation,inhibiting apoptosis,and increasing the secretion of extracellular matrix.Among them,umbilical cord mesenchymal stem cell exosomes had stronger cartilage repair activity.(4)After the treatment of both exosomes,the macroscopic performance and OARSI score of articular cartilage were better than those of the control group,and the inflammatory factors in the joint fluid were decreased,among which the efficacy of umbilical cord mesenchymal stem cell exosomes was better than that of bone marrow mesenchymal stem cell exosomes.(5)Results suggest that the yield and purity of umbilical cord mesenchymal stem cell exosomes are higher than that of bone marrow mesenchymal stem cell exosomes,and the therapeutic effect of umbilical cord mesenchymal stem cell exosomes on the treatment of osteoarthritis is better than that of bone marrow mesenchymal stem cell exosomes.Therefore,umbilical cord mesenchymal stem cell exosome is more suitable as a new strategy for osteoarthritis.