腺相关病毒基因修饰后间充质干细胞来源的外泌体免疫调节功能的研究  

The immunoregulatory functions of exosome derived from mesenchymal stem cells that are genetically modified by adeno-associated virus

作　　者：刘丽 邵文威 李成文 冯四洲 裴晓磊 Liu Li;Shao Wenwei;Li Chengwen;Feng Sizhou;Pei Xiaolei(State Key Laboratory of Experimental Hematology,National Clinical Research Center for Blood Diseases,Institute of Hematology&Blood Diseases Hospital,Chinese Academy of Medical Sciences&Peking Union Medical College,Tianjin 300020,China;Academy of Medical Engineering and Translational Medicine,Medical College,Tianjin University,Tianjin 300072,China;Gene Therapy Center,University of North Carolina at Chapel Hill,NC 27599,USA)

机构地区：[1]中国医学科学院血液病医院、北京协和医学院(中国医学科学院血液学研究所),实验血液学国家重点实验室,国家血液系统疾病临床医学研究中心,天津300020 [2]天津大学,医学工程与转化医学研究院,300072 [3]北卡罗莱纳大学(教堂山),医学院基因治疗中心,美国北卡罗莱纳州

出　　处：《中华血液学杂志》2021年第6期452-458,共7页Chinese Journal of Hematology

基　　金：国家自然科学基金面上项目(8207072323);中国医学科学院临床与转化医学研究基金(2020-I2M-C&T-B-088)。

摘　　要：目的验证天然状态间充质干细胞来源的外泌体(MSC-exosome)治疗小鼠急性移植物抗宿主病(GVHD)的效果和可能机制,探索并建立定向基因修饰MSC-exosome的方法并验证修饰后MSC-exosome的功能。方法构建小鼠急性GVHD模型,观察比较不同剂量MSC-exosome组和MSC组的生理指标评分、生存和体重减低程度;进而通过体外T细胞活化实验和体内OVA抗原特异性T细胞活化实验检测比较组间活化T细胞的增殖水平。构建重组表达载体,获得携带PD-L1和PD-L1-ITGB1的AAV2YF3突变体,进而感染人MSC,分离获得其外泌体。检测比较天然状态和修饰后MSC-exosome在体外和体内对活化T细胞的增殖水平和调节性T细胞(Treg)比例的影响。结果①MSC-exosome(300μg×3次)和小鼠MSC(1×10^(6)×3次)均能够有效改善急性GVHD小鼠的生理指标评分、生存和体重减低程度。②相比IL-2对照组,10、25、50μg人MSC-exosome和1×10^(6)人MSC体外共孵育处理均能够抑制活化T细胞的增殖,增殖比例分别为86.0%(IL-2)、40.0%、39.6%、42.8%和41.0%;相比PBS对照组,50、100、200μg小鼠MSC-exosome和1×10^(6)小鼠MSC在体内均能够抑制抗原特异性活化的OT-1细胞的增殖,增殖比例分别为42.6%、33.1%、14.2%、10.6%和14.6%。③携带PD-L1和PD-L1-ITGB1的AAV2YF3突变体定向修饰人MSC-exosome的表达率分别超过40%和60%。④相比天然状态,PD-L1和PD-L1-ITGB1定向修饰后MSC-exosome在体内对抗原特异性活化的OT-1细胞具有更好的增殖抑制效果;在体外亦能明显抑制活化T细胞的增殖,并诱导提高Treg的比例。结论MSC-exosome具有与MSC相似的免疫调节作用。经过PD-L1和PD-L1-ITGB1修饰后的MSC-exosome能够有效抑制活化T细胞的增殖,并且能够诱导提高Treg的比例。Objectives To verify the effects and mechanisms of natural MSC-exosome in treating acute GVHD in mice,explore and establish a method for targeted modification of MSC-exosome,and verify the functions of the modified MSC-exosome.Methods In different doses of MSC-exosome groups and MSC group,weight loss in acute GVHD mice was observed;then the proliferation levels of activated T cells were measured through T cell activation experiment in vitro and OVA antigen-specific T cell activation experiment in vivo.AAV2YF3 mutants carrying PD-L1 and PD-L1-ITGB1 were obtained after the construction of recombinant expression vectors and were then applied to infect human MSC to modify their exosome.The immunoregulatory functions of the modified MSC-exosome were measured with the abovementioned methods.Results①Mouse MSC-exosome(300μg×3 times)and MSC(1×10^(6)×3 times)effectively alleviated the weight loss in acute GVHD mice.②Compared with IL-2,10,25 and 50μg human MSC-exosome inhibited the proliferation of activated T cells in vitro,respectively,86.0%(IL-2),40.0%,39.6%,and 42.8%;compared with PBS,50,100 and 200μg mouse MSC-exosome inhibited the proliferation of antigen-specific activated OT-1 cells in vivo,respectively,42.6%,33.1%,14.2%,and 10.6%.③After the infection of AAV2YF3 mutant carrying PD-L1 or PD-L1-ITGB1,the positive proportion of MSC-exosome exceeds 40%and 60%,respectively.④Compared with the natural state,MSC-exosome modified by PD-L1 or PD-L1-ITGB1 showed better proliferation inhibitory effect in vivo and increased the proportion of Treg cells in vitro.Conclusions MSC-exosome exhibited similar immunomodulatory effects with MSC.MSC-exosome after PD-L1 and PD-L1-ITGB1-targeted modification effectively inhibited the proliferation of activated T cells and increased the proportion of Treg cells.

关 键 词：间充质干细胞 外泌体 移植物抗宿主病 腺相关病毒 基因修饰 

分 类 号：R457.7[医药卫生—治疗学]