高表达软骨调素1骨髓间充质干细胞来源外泌体促进骨关节炎软骨细胞的增殖

Exosomes derived from bone marrow mesenchymal stem cells with chondromodulin-1 overexpression affect the proliferation of chondrocytes in osteoarthritis

背景:软骨调素1(chondromodulin-1,Chm-1)在正常软骨中大量表达,具有抑制血管生成、阻止软骨细胞肥大、缓解骨关节炎发展的功能,在骨关节炎发生和发展中发挥着重要作用。目的:探讨高表达Chm-1骨髓间充质干细胞来源外泌体对骨关节炎环境中软骨细胞增殖的影响。方法:分离大鼠骨髓间充质干细胞并进行培养及鉴定;Chm-1慢病毒和空载慢病毒转染骨髓间充质干细胞,超高速离心法提取细胞培养上清液中外泌体(Chm-1-Exos和NC-Exos)。白细胞介素1β作用软骨细胞24 h构建体外骨关节炎模型,随后分别用2种外泌体处理7 d,将PBS处理的骨关节炎软骨细胞作为对照组。共聚焦显微镜观察骨关节炎软骨细胞对外泌体的摄取情况;CCK-8评估各组骨关节炎软骨细胞增殖能力;Western blot检测2种外泌体中Chm-1的表达;Western blot和RT-qPCR方法分别检测各组骨关节炎软骨细胞中Ⅱ型胶原、SOX9、AGG蛋白和mRNA的表达。结果与结论:(1)骨髓间充质干细胞呈典型纺锤状形态且具备三系分化能力;(2)带有绿色荧光的慢病毒成功转染骨髓间充质干细胞;(3)外泌体直径在40-150 nm之间,且均表达其特异性标志蛋白CD63、CD81、TSG101,能够被骨关节炎软骨细胞摄取;(4)相比于NC-Exos,Chm-1-Exos中Chm-1的表达更高(P<0.05);(5)NC-Exos和Chm-1-Exos分别作用骨关节炎软骨细胞24 h后,Chm-1-Exos组骨关节炎软骨细胞活性更强;(6)NC-Exos和Chm-1-Exos分别作用骨关节炎软骨细胞7 d后,Chm-1-Exos组的Ⅱ型胶原、SOX9、AGG蛋白和mRNA表达更高(P<0.05);(7)上述实验结果表明,Chm-1-Exos能够显著促进骨关节炎软骨细胞的增殖。

BACKGROUND:Chondromodulin-1(Chm-1),which is highly expressed in normal cartilage,can inhibit angiogenesis,prevent chondrocyte hypertrophy and alleviate the development of osteoarthritis.Chm-1 plays an important role in the occurrence and development of osteoarthritis.OBJECTIVE:To investigate the effect of exosomes(Exos)derived from bone marrow mesenchymal stem cells with high expression of Chm-1 on the proliferation of chondrocytes in osteoarthritis.METHODS:Rat bone marrow mesenchymal stem cells were isolated,cultured and identified.Bone marrow mesenchymal stem cells were transfected with Chm-1 lentivirus and no-load lentivirus.NC-Exos and Chm-1-Exos in the supernatant of cell culture were extracted by ultra-high speed centrifugation.The model of osteoarthritis in vitro was established by treating chondrocytes with interleukin-1βfor 24 hours.Then osteoarthritis chondrocytes were treated with NC-Exos and Chm-1-Exos for 7 days,respectively,and PBS-treating osteoarthritis chondrocytes were used as a control group.The Exos uptake of osteoarthritis chondrocytes was observed by a confocal microscope.The proliferation of osteoarthritis chondrocytes was evaluated by CCK-8 assay.Expression levels of Chm-1 in NC-Exos and Chm-1-Exos were detected by western blot assay.The protein and mRNA expression levels of ColⅡ,SOX9 and AGG in osteoarthritis chondrocytes were detected by western blot assay and RT-qPCR,respectively.RESULTS AND CONCLUSION:(1)Bone marrow mesenchymal stem cells showed a typical fusiform shape and had the ability to differentiate into three lines.(2)Lentivirus with green fluorescence was successfully transfected into bone marrow mesenchymal stem cells.(3)Exos,whose size was between 40-150 nm,could be absorbed by osteoarthritis chondrocytes and expressed their specific marker proteins CD63,CD81 and TSG101.(4)Compared with NC-Exos,Chm-1-Exos expressed higher proteins of Chm-1(P<0.05).(5)At 24 hours after treatment,compared with the NC-Exos group,the Chm-1-Exos group presented higher cell viability.(6)At 7 days after treatment,compared with the NC-Exos group,the Chm-1-Exos group showed higher protein and mRNA expression levels of ColⅡ,SOX9 and AGG(P<0.05).(7)The above experimental results have shown that Chm-1-Exos can promote the proliferation of osteoarthritis chondrocytes.