功能化外泌体对实验性自身免疫性脑脊髓炎小鼠治疗的研究  

Study of functionalized exosomes for treating experimental autoimmune encephalomyelitis in mice

作　　者：陈傲 邱帅 李悦 尹海芳 CHEN Ao;QIU Shuai;LI Yue;YIN Haifang(Department of Cell Biology,School of Basic Medical Sciences,Tianjin Medical University,Tianjin 300070,China)

机构地区：[1]天津医科大学基础医学院细胞生物学系,天津300070

出　　处：《天津医科大学学报》2024年第3期212-217,共6页Journal of Tianjin Medical University

基　　金：中央支持地方高校发展基金(116003/XK010302)。

摘　　要：目的:探究负载髓鞘少突胶质细胞糖蛋白(MOG)的293F细胞来源的外泌体(EXO)对实验性自身免疫性脑脊髓炎(EAE)小鼠的治疗效果。方法:利用Western印迹、透射电子显微镜以及Nanosight对超速离心分离的293F细胞上清EXO进行表征;利用流式细胞术和共聚焦显微镜检测细胞摄取EXO的生物学功能;利用EXO特异性锚定肽EXP将MOG抗原肽修饰于EXO表面,流式细胞术检测EXO与MOG抗原肽的结合效率(EXO_(MOG));活体成像检测EXO_(MOG)的体内分布;利用小鼠的临床评分、体重变化以及收样时小鼠脾细胞中调节性T细胞变化和脊髓染色,评估EXO_(MOG)对EAE小鼠的治疗效果;利用组织形态学染色检测EXO_(MOG)治疗对各组小鼠不良反应。结果:超速离心可成功分离EXO,且分离的EXO具有被细胞摄取的生物学功能;EXP外泌体锚定肽介导MOG抗原肽高效负载于EXO表面,且不影响EXO的体内分布;在EAE小鼠上测试结果显示:与未治疗组和MOG组相比,EXO_(MOG)组小鼠治疗后第24天的体重显著增加(P<0.05),行为学评分显著降低(P<0.05),且治疗后小鼠脾脏中CD4^(+)CD25^(+)Treg细胞显著增高(P<0.05);病理染色结果表明:EXO_(MOG)组小鼠的各组织器官未见明显病理学改变。结论:EXO_(MOG)能够有效缓解小鼠EAE疾病进展且未检测到相关不良反应。Objective:To explore the therapeutic effect of exosomes(EXO)derived from 293F cells loaded with myelin oligodendrocyte glycoprotein(MOG)on experimental autoimmune encephalomyelitis(EAE)mice.Methods:Characterization of EXO from the culture supernatant of 293F cells separated by ultracentrifugation using Western blotting,transmission electron microscopy,and nano-sight.Flow cytometry and confocal microscopy were used to detect the biological function of cells taking up EXO.MOG antigen peptides were loaded on the surface of EXO via exosomal anchor peptide-EXP.Flow cytometry was used to detect the binding efficiency of EXO and MOG antigen peptide(EXO_(MOG))and small animal imaging was applied to examine the distribution of EXO_(MOG).The therapeutic effect of EXO_(MOG)on EAE mice was evaluated by the changes of clinical score,weight,regulatory T cells in spleen cells and spinal cord staining at the time of sample collection.The toxic of EXO_(MOG)on mice in each group were detected by tissue morphology staining.Results:EXO could be successfully separated by ultracentrifugation,and the EXO had the biological function of being taken up by cells.The anchor peptide of EXP exosomes mediated the efficient loading of MOG antigen peptides on the surface of EXO without altering the in vivo distribution of EXO.The test results on EAE mice showed that compared to the untreated group and MOG alone group,EXO_(MOG)treatment significantly increased the weight on the 24th day after induction(P<0.05).Behavioral scores significantly declined in EAE mice treated with EXO_(MOG)compared with untreated controls(P<0.05).The immuno-tolerogenic CD4^(+)CD25^(+)Treg cells in the spleen of mice significantly increased after treatment(P<0.05).The pathological examination revealed that there were no significant pathological changes in the tissues and organs of the EXO_(MOG)group mice.Conclusion:EXO_(MOG)are able to effectively prevent the progression of EAE disease in mice without any drug-associated toxicity.

关 键 词：多发性硬化 实验性自身免疫性脑脊髓炎 外泌体 293F悬浮细胞 髓鞘少突胶质细胞糖蛋白 

分 类 号：Q291[生物学—细胞生物学]