骨髓间充质干细胞来源外泌体保护软骨细胞延缓骨关节炎的发生发展

Bone marrow mesenchymal stem cell derived exosomes delay the occurrence and development of osteoarthritis through cartilage protection

背景:骨关节炎是最常见的关节退行性疾病,目前骨髓间充质干细胞已应用于骨关节炎治疗中,但与骨髓间充质干细胞相比,骨髓间充质干细胞来源外泌体移植具有更多优势,例如非免疫原性、非致瘤性以及储存和运输方便。目的:探索骨髓间充质干细胞来源外泌体对骨关节炎的保护作用。方法:①提取SD大鼠骨髓间充质干细胞并通过细胞形态及流式细胞学进行鉴定,利用超速离心法提取细胞上清液中的外泌体并通过透射电镜、粒径及western blot进行鉴定;②提取原代乳鼠肋软骨细胞,与荧光标记外泌体共培养12 h,观察软骨细胞吞噬情况。体外通过白细胞介素1β诱导软骨细胞损伤,加入含有50μg外泌体的PBS溶液100μL干预24 h,采用免疫荧光检测基质金属蛋白酶13及Ⅱ型胶原纤维α1蛋白表达,评估外泌体对于损伤软骨细胞的保护作用;③体内通过碘乙酸诱导大鼠骨关节炎模型,关节腔内注射外泌体,通过苏木精-伊红染色及番红-固绿染色观察骨关节炎关节结构变化,通过免疫组织化学染色检测基质金属蛋白酶13及Ⅱ型胶原纤维α1蛋白表达,评估外泌体对体内软骨损伤的保护作用。结果与结论:①原代骨髓间充质干细胞呈现典型的梭形,放射状排列,表达CD73和CD105而几乎不表达CD45、CD34和CD3;透射电镜显示外泌体呈现典型茶托样形态,粒径小于100 nm,ALIX及HRS蛋白呈阳性表达;②软骨细胞内可观察到典型的红染颗粒证实外泌体可以被软骨细胞摄取,同时外泌体可以促进软骨细胞Ⅱ型胶原纤维α1蛋白表达及抑制基质金属蛋白酶13表达,证实外泌体可以减弱白细胞介素1β对软骨细胞的损伤作用;③外泌体可以促进损伤关节软骨形态恢复及Ⅱ型胶原纤维α1表达,抑制基质金属蛋白酶13表达,同样证实外泌体可以缓解碘乙酸对骨关节软骨的破坏作用;④上述结果表明,骨髓间充质干细胞外泌体通过软骨保护机制延缓骨关节炎的发生与发展。

BACKGROUND:Osteoarthritis is the most common joint degenerative disease.At present,bone marrow mesenchymal stem cells have been used in the treatment of osteoarthritis.However,compared with bone marrow mesenchymal stem cells,bone marrow mesenchymal stem cell derived exosome transplantation has more advantages,such as non-immunogenicity,non-tumorigenicity,convenient storage and transportation.OBJECTIVE:To explore the protective effect of bone marrow mesenchymal stem cell exosomes on osteoarthritis.METHODS:(1)SD rat bone marrow mesenchymal stem cells were extracted and identified by cell morphology and flow cytometry.Exosomes in the cell supernatant were extracted by ultracentrifugation and identified by transmission electron microscopy,particle size and western blot assay.(2)Primary costal chondrocytes were extracted from suckling rats and cocultured with fluorescently labeled exosomes for 12 hours.The phagocytosis of chondrocytes was observed.In vitro chondrocyte damage was induced by interleukin-1β.PBS(100μL)containing 50μg exosomes was added for 24 hours.The expression of matrix metalloproteinase-13 and type II collagen fiberα1 protein was detected by immunofluorescence to evaluate the protective effect of exosomes on injured chondrocytes.(3)The rat model of osteoarthritis was induced by iodoacetic acid in vivo.Exosomes were injected into the joint cavity,and the changes of joint structure of osteoarthritis were observed by hematoxylin-eosin staining and safrane-fast green staining.The expression of matrix metalloproteinase-13 and type II collagen fiberα1 protein was measured by immunohistochemical staining to evaluate the protective effect of exosomes on cartilage in vivo.RESULTS AND CONCLUSION:(1)The extracted primary cells showed a typical fusiform shape and arranged radially.The extracted cells highly expressed CD73 and CD105,but slightly expressed CD45,CD34 and CD3.Transmission electron microscopy showed that the obtained particles showed a typical saucer-like morphology.The particle size was less than 100 nm.Meanwhile,nanoparticles showed positive expression of ALIX and HRS protein.(2)Typical red-stained particles could be observed in chondrocytes,which confirms that exosomes could be taken up by chondrocytes,and exosomes could promote chondrocyte type II collagen fiberα1 protein expression,but inhibit the expression of matrix metalloproteinase-13,which confirmed that exosomes could attenuate the damage effect of interleukin-1βon chondrocytes.(3)Exosomes could promote the morphological recovery of damaged articular cartilage and the up-regulate type II collagen fiberα1 expression,while inhibited the expression of matrix metalloproteinase-13,which also confirmed that exosomes can alleviate the effects of iodoacetic acid on articular cartilage damage.(4)Above findings results indicate that bone marrow mesenchymal stem cell exosomes delay the occurrence and development of osteoarthritis through a chondroprotective mechanism.