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

摘要

目的:探讨突变型低氧诱导因子1α(HIF-1α)修饰骨髓间充质干细胞(BMSCs)分泌的外泌体对软骨细胞的保护作用,阐明其联合软骨再生支架促进晚期软骨缺损修复的可能机制.方法:采用超速离心法从野生型 HIF-1α和突变型 HIF-1α修饰的BMSCs中分别提取外泌体(BMSCs-ExoWT与BMSCs-ExoMU),同时对其进行鉴定.在体外,采用白细胞介素1β(IL-1β)诱导软骨细胞发生炎症反应,分别将等量 PBS、BMSCs-ExoWT (80 mg·L-1)和BMSCs-ExoMU(80 mg·L-1)分别与炎症反应下的软骨细胞共培养,实验分为空白组、炎症组、BMSCs-ExoWT组和 BMSCs-ExoMU组,利用 Hoechst33342染色检测各组软骨细胞凋亡小体数目;应用Western blotting法检测各组软骨细胞中 AKT/p-AKT、ERK/p-ERK和 p38/p-p38表达水平.12只新西兰兔随机分为4组,建立兔膝关节软骨缺损模型,分别将等量的生理盐水、支架+生理盐水、支架+BMSCs-ExoWT和支架+BMSCs-ExoMU作用于4组兔软骨缺损处.术后6周取材,通过大体观察、苏木素-伊红(HE)染色和蕃红 O-固绿染色观察和比较各组软骨缺损的修复效果.结果:成功提取并鉴定 BMSCs-ExoWT与 BMSCs-ExoMU,电镜观察外泌体形态为近圆形,直径为40~100 nm;Western blotting法显示两者分别表达特异性蛋白 CD63和CD81.在体外实验中,炎症环境下BMSCs-ExoMU组软骨细胞凋亡小体数目低于炎症组和BMSCs-ExoWT组(P<0.01);Western blotting法,BMSCs-ExoMU组和BMSCs-ExoWT组软骨细胞中 p-ERK1和 p-ERK2水平低于炎症组(P<0.05),p-AKT和p-p38水平高于炎症组(P<0.05);且BMSCs-ExoMU的作用强于BMSCs-ExoWT(P<0.05).在兔膝关节晚期软骨缺损模型中,支架+BMSCs-ExoMU组缺损处修复效果优于空白组、支架组和支架+BMSCs-ExoWT组.结论:软骨支架与BMSCs-ExoMU共同作用于软骨缺损处可促进缺损修复.%Objective:To explore the protective effect of exosomes secreted from bone mesenchymal stem cells (BMSCs)modified by hypoxia inducible factor-1α(HIF-1α)on the chondrocytes,and to elucidate the possible mechanism of its combination with cartilage regenerated scaffolds in promoting the repair of advanced cartilage defects.Methods:The exosomes(BMSCs-ExoWTand BMSCs-ExoMU)were extracted from the BMSCs modified by wild type of HIF-1α and mutant type of HIF-1α by ultracentrifugation method and identified in the meantime.In vitro the inflammatory response of chondrocytes were induced by interleukin-1β(IL-1β),the same amount of PBS, BMSCs-ExoWT(80 μg · mL-1),BMSCs-ExoMU(80 μg · mL-1)were respectively cultivated with the chondrocytes under the inflammatory reaction and blank group,inflammation group,BMSCs-ExoWTgroup and BMSCs-ExoMUgroup were set up;Hoechst33342 staining was used to detect the number of apoptotic bodies of chondrocytes in various groups.The Western blotting method was used to detect the expression levels of AKT/p-AKT,ERK/p-ERK and p38/p-p38 in the chondrocytes in various groups.Twelve New Zealand white rabbits were randomly divided into 4 groups and the models of rabbit knee cartilage defects were consructed;the equal volume of physiological saline,scaffold +physiological saline,scaffold +BMSCs-ExoWTand scaffold +BMSCs-ExoMUwere respectively injected into the cartilage defects of rabbits.Six weeks after operation,gross conference, HE and safranin O staining were used to observe and compare the repair effects of cartilage defects in each group. Results:BMSCs-ExoWTand BMSCs-ExoMUwere successfully extracted and identified,and the exosomes were observed to be nearly circular with diameter of about 40-100 nm;the Western blotting results showed that they expressed special proteins CD63 and CD81,respectively.In vitro,the number of apoptotic bodies of chondrocytes in BMSCs-ExoMUgroup was lower than those in inflammation group and BMSCs-ExoWTgroup(P<0.01).The Western blotting results revealed that the expression levels of p-ERK1/2 in BMSCs-ExoMUand BMSCs-ExoWT groups were lower than that in inflammation group(P<0.05);the expression levels of p-AKT and p-p38 were higher(P<0.05);the effect in BMSCs-ExoMUgroup was stronger than BMSCs-ExoWTgroup,and the difference was statistically significant(P<0.05).In the advanced cartilage defect models of rabbit knee joint,the repair effect in scaffold+ BMSCs-ExoMUgroup was better than those in blank group,scaffold group and scaffold+BMSCs-ExoWTgroup.Conclusion:Cartilage scaffold combined with BMSCs-ExoMUcan promote the repair of cartilage defects.