温敏型壳聚糖水凝胶包封外泌体在缺血性疾病中的应用  

Application of temperature-sensitive chitosan hydrogel encapsulated exosomes in ischemic diseases

作　　者：刘冯 张瑜 王燕丽 骆威 韩超珊 李杨欣 Liu Feng;Zhang Yu;Wang Yanli;Luo Wei;Han Chaoshan;Li Yangxin(Institute of Cardiovascular Diseases,Medical College of Soochow University,Suzhou 215000,Jiangsu Province,China;Department of Cardiovascular Surgery,First Affiliated Hospital of Soochow University,Suzhou 215006,Jiangsu Province,China)

机构地区：[1]苏州大学医学部心血管病研究所,江苏省苏州市215000 [2]苏州大学附属第一医院心脏大血管外科,江苏省苏州市215006

出　　处：《中国组织工程研究》2021年第16期2479-2487,共9页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金重大研究计划项目(91849122),项目负责人:李杨欣;国家自然科学基金面上项目(81870194),项目负责人:李杨欣;江苏省“六大人才高峰”创新团队项目(BU 24600117),项目负责人:李杨欣。

摘　　要：背景:基于间充质干细胞来源的外泌体可减少心肌缺血和再灌注损伤,但存在半衰期短、清除速度快和靶向性低等问题。目的:对外泌体进行修饰和改造,并用温敏型水凝胶包封外泌体,以提高外泌体在体内的驻留率,改善其治疗效果。方法:利用细胞转染的方法敲低脐带间充质干细胞来源外泌体中的piR823,检测敲低piR823的外泌体对C2C12细胞增殖与凋亡的影响。制备壳聚糖/β-甘油磷酸钠温敏型水凝胶,将其与外泌体直接混合制备包封外泌体的壳聚糖/β-甘油磷酸钠温敏型水凝胶,检测包封外泌体后水凝胶的成胶性能、流变学及体外缓释性能。取30只C57BL/6J小鼠,建立后肢缺血模型,随机分5组干预:A组腓肠肌注射PBS,B组注射壳聚糖/β-甘油磷酸钠温敏型水凝胶,C组注射包封外泌体的壳聚糖/β-甘油磷酸钠温敏型水凝胶,D组注射外泌体,E组注射敲低piR823的外泌体,分别进行肢体功能与恢复、血流、四肢抓力、运动耐力及肌肉质量再生检测。结果与结论:①敲低piR823的外泌体抑制C2C12细胞的增殖;正常外泌体抑制过氧化氢诱导的C2C12细胞凋亡,敲低piR823后外泌体的抑制细胞凋亡作用减弱;②包封外泌体的水凝胶具有成胶性能,但成胶时间缩短,其可缓慢持续释放外泌体达30 d以上;③后肢缺血28 d后,C组的左肢血流恢复优于B、D组(P<0.05),D组优于E组(P<0.05);C组的四肢抓力、运动耐力跑步时间和距离优于D、B组(P<0.05),D组优于E组(P<0.05);C组肌肉再生情况优于B、D组(P<0.05),D组优于E组(P<0.05);④结果表明,以壳聚糖/β-甘油磷酸钠温敏型水凝胶包封外泌体延长了外泌体在体内的滞留时间,明显增强了血流灌注及缺血后组织功能的恢复,治疗效果更显著。BACKGROUND:Exosomes derived from mesenchymal stem cells can reduce myocardial ischemia and reperfusion injury,but there are some problems such as short half-life,fast clearance and low targeting.OBJECTIVE:To modify and encapsulate exosomes with temperature-sensitive chitosan hydrogel to increase the retention rate of exosomes in the body,and to achieve better therapeutic effect.METHODS:Cell transfection method was used to knock down piR823 in exosomes derived from umbilical cord mesenchymal stem cells,and the effect of knockdown of piR823 exosomes on the proliferation and apoptosis of C2 C12 cells was detected.Chitosan/β-sodium glycerophosphate temperature-sensitive hydrogel was prepared and mixed directly with exosomes to prepare chitosan/β-sodium glycerophosphate temperature-sensitive hydrogel encapsulated with exosomes.The gel-forming properties,rheology and in vitro sustained release properties of the hydrogel after encapsulation of exosomes were tested.Thirty C57 BL/6 J mice were taken to establish hind limb ischemia models,and randomly divided into five groups.The gastrocnemius of group A was injected with PBS;group B was injected with chitosan/β-sodium glycerophosphate temperature-sensitive hydrogel;and group C was injected with exosomes-encapsulated chitosan/β-sodium glycerophosphate temperature-sensitive hydrogel;group D was injected with exosomes;group E was injected with exosomes knocking down piR823.Limb function and recovery,blood flow,grip strength,exercise endurance and muscle regeneration were detected in each group.RESULTS AND CONCLUSION:(1)Exosomes knocking down piR823 inhibited the proliferation of C2 C12 cells;normal exosomes inhibited hydrogen peroxide-induced apoptosis of C2 C12 cells.The inhibitory effect of exosomes on apoptosis was weakened after piR823 was knocked down.(2)The hydrogel encapsulating exosomes had gel-forming properties,but the gel-forming time was shortened,and it could slowly and continuously release exosomes for more than 30 days.(3)After 28 days of hindlimb ischemia

关 键 词：材料 外泌体 壳聚糖 温敏型水凝胶 间充质干细胞 后肢缺血 动物模型 PIRNA 

分 类 号：R459.9[医药卫生—治疗学] R318.08[医药卫生—临床医学]