人脐带间充质干细胞来源外泌体对脑瘫小鼠神经功能损伤的保护作用  

Neuroprotective effects of exosomes derived from human umbilical cord mesenchymal stem cells in cerebral palsy mice

作　　者：陈星星 赛依帕[1] 胡筱霞 王三萍[1] 罗璇[1] 刘璟[1] CHEN Xingxing;SAI Yipa;HU Xiaoxia;WANG Sanping;LUO Xuan;LIU Jing(Department of Pediatrics,People's Hospital of Gansu Province,the First Clinical Medical College of Gansu University of Chinese Medicine,Lanzhou 730000,China)

机构地区：[1]甘肃省人民医院甘肃中医药大学第一临床医学院儿科,甘肃兰州730000

出　　处：《基础医学与临床》2023年第8期1201-1207,共7页Basic and Clinical Medicine

基　　金：甘肃省自然科学基金(22JR5RA689);甘肃省青年科技基金计划(22JR5RA701);甘肃省人民医院院内科研基金(20GSSY1-13);甘肃省外科肿瘤分子诊断与精准治疗重点实验室项目(GSSZDSYS2020-10)。

摘　　要：目的探讨人脐带间充质干细胞(HUCMSCs)外泌体对脑性瘫痪(简称脑瘫,CP)小鼠神经功能损伤的保护作用及其机制。方法HUCMSCs培养、鉴定后提取其外泌体。透射电镜(TEM)、纳米颗粒追踪分析仪(NTA)和Western blot鉴定外泌体;将小鼠随机分为假手术组(sham组,n=6)、模型组[CP组,n=8,单侧颈总动脉结扎联合脂多糖(LPS)感染后缺氧诱导的方法建立CP模型]、模型-外泌体组(CP-Exo组,n=8,尾静脉注射125μg外泌体)。Longa评分、网格试验和爬杆试验评估小鼠的神经运动功能、四肢抓握能力和平衡能力;苏木精-伊红(HE)染色光镜下观察脑组织病理损伤,透射电镜观察神经元突触结构,甲苯胺蓝染色检测神经元存活数目,Western blot检测突触相关蛋白突触后致密物95(PSD-95)、突触小泡蛋白(SYP)的表达。结果HUCMSCs外泌体为形态完整、球形、双层膜囊泡结构,平均粒径123 nm,外泌体TSG101蛋白表达阳性;Longa评分CP组小鼠得分较sham组显著增加(P<0.01);CP-Exo组评分显著低于CP组(P<0.05);与CP组相比,CP-Exo组未见斑片状坏死且白质区神经纤维脱髓鞘发生率明显减低(P<0.01);脑组织结构中sham组突触结构相对正常,另外两组样本均存在不同程度的病变,其病变的大致趋势为:CP组>CP-Exo组>sham组;CP组脑组织海马区锥体细胞尼氏小体数量显著低于sham组(P<0.01),CP-Exo组脑组织海马区锥体细胞尼氏小体数量显著高于CP组(P<0.01);而CP组PSD-95和SYP表达水平显著低于sham组(P<0.01),CP-Exo组PSD-95和SYP表达显著高于CP组(P<0.01)。结论HUCMSCs外泌体可促进脑瘫小鼠神经功能的恢复,可能通过增加PSD-95和SYP蛋白的表达减轻神经突触的损伤,保护神经元,从而达到改善脑瘫小鼠运动功能的目的。Objective To investigate the neuroprotective effects of exosomes derived from human umbilical cord mesenchymal stem cells(HUCMSCs)in cerebral palsy(CP)mice.Methods The exosomes from HUCMSCs were verified by transmission electron microscopy(TEM),nanoparticle tracking analyzer(NTA)and Western blot.The animals were randomly divided as sham operation group(sham group,n=8),model group[CP group,n=8,hypoxic induction after unilateral common carotid artery ligation combined with lipopolysaccharide(LPS)infection]and model exosome group(CP-Exo group,n=8,125μg exosomes was injected intravenously).Neuromotor function,the ability of grasping and balance were evaluated by Longa score,grid test and pole climbing test.The pathological lesion in brain tissue was observed with hematoxylin-eosin(HE)staining,the synaptic structure of neurons was observed by TEM,the survival of neurons was detected by toluidine blue staining,and the expression levels of PSD-95 and synaptophysin(SYP)were detected by Western blot.Results The exosomes isolated were spherical and double-membrane vesicles with an average particle size of 123nm,which expressed TSG101 protein.In animal behavior tests,Longa score in CP group was significantly higher than sham group(P<0.01)and CP-Exo group(P<0.05).There was no plaque necrosis found in exosome group and the incidence of nerve fiber demyelination in the white matter area was significantly reduced than in the CP group(P<0.01).The synaptic structure of the sham group was relatively normal,while the other two groups showed varying degrees of pathological changes.The general trend of pathological changes was CP group>CP-Exo group>sham group.The number of Nissl bodies in hippocampal pyramidal cells in CP group was significantly less than that in sham group(P<0.01),however,which was also less than that in CP-Exo group(P<0.01).The expression of PSD-95 and SYP in CP group was significantly lower than that in sham group(P<0.01),and the levels PSD-95 and SYP in CP-Exo group was significantly higher than those in CP grou

关 键 词：脑性瘫痪 外泌体 脐带间充质干细胞 

分 类 号：R742[医药卫生—神经病学与精神病学] R364[医药卫生—临床医学]