吉林大学学报(医学版) ›› 2018, Vol. 44 ›› Issue (02): 216-222.doi: 10.13481/j.1671-587x.20180203

• 基础研究 • 上一篇    下一篇

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

田大川1, 李海乐2, 肖大伟1, 周山健1, 苏永蔚1, 刘丹平1, 綦惠3   

  1. 1. 锦州医科大学附属第一医院运动与关节科, 辽宁 锦州 121000;
    2. 河南省漯河市 中心医院创伤骨科, 河南 漯河 462000;
    3. 北京市创伤骨科研究所, 北京 100035
  • 收稿日期:2017-06-17 出版日期:2018-03-28 发布日期:2018-03-30
  • 通讯作者: 刘丹平,教授,硕士研究生导师(Tel:0416-4197673,E-mail:liudanping2009@sohu.com) E-mail:liudanping2009@sohu.com
  • 作者简介:田大川(1991-),男,安徽省蚌埠市人,在读医学硕士,主要从事关节软骨缺损修复方面的研究。
  • 基金资助:
    国家自然科学基金资助课题(81572140);北京市自然科学基金面上项目资助课题(7172036)

Promotion effect of cartilage regenerated scaffolds combined with exosomes derived from mutant type of HIF-1α modified BMSCs in repairing advanced cartilage defects

TIAN Dachuan1, LI Haile2, XIAO Dawei1, ZHOU Shanjian1, SU Yongwei1, LIU Danping1, QI Hui3   

  1. 1. Department of Orthopedics, First Affiliated Hospital, Jinzhou Medical University, Jinzhou 121000, China;
    2. Department of Traumatologyand Orthopedics, Central Hospital of Luohe City, Henan Province, Luohe 462000, China;
    3. Beijing Institute of Traumatology and Orthopedics, Beijing 100035, China
  • Received:2017-06-17 Online:2018-03-28 Published:2018-03-30

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40~100nm;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-ExoWTP<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。

关键词: 骨髓间充质干细胞, 软骨再生支架, 外泌体, 软骨缺损, 软骨细胞, 白细胞介素1β

Abstract: 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-ExoWT and 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-ExoWT group and BMSCs-ExoMU group 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-ExoWT and scaffold +BMSCs-ExoMU were 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-ExoWT and BMSCs-ExoMU were 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-ExoMU group was lower than those in inflammation group and BMSCs-ExoWT group(P<0.01).The Western blotting results revealed that the expression levels of p-ERK1/2 in BMSCs-ExoMU and 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-ExoMU group was stronger than BMSCs-ExoWT group,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-ExoMU group was better than those in blank group,scaffold group and scaffold+BMSCs-ExoWT group. Conclusion: Cartilage scaffold combined with BMSCs-ExoMU can promote the repair of cartilage defects.

Key words: cartilage defect, cartilage regeneration scaffold, bone meserchymal stem cells, chondrocyte, interleukin-1β, exosomes

中图分类号: 

  • R684