微血管内皮细胞来源的外泌体对血管性痴呆模型小鼠脑血管形成及神经损伤的作用研究  

Effect of Exosomes Derived from Microvascular Endothelial Cells on Cerebrovascular Formation and Neurological Injury in Vascular Dementia Model

作　　者：林东晓 陈贞君 李华[1] 黄星华 LIN Dongxiao;CHEN Zhenjun;LI Hua;HUANG Xinghua(Cangshan Campus,The 900th Hospital of Joint Logistics Support Force,Fuzhou 350003,Fujian,China)

机构地区：[1]中国人民解放军联勤保障部队第九○○医院仓山院区,福州350003 [2]福建省立医院临床医学实验研究中心

出　　处：《中西医结合心脑血管病杂志》2024年第8期1409-1415,共7页Chinese Journal of Integrative Medicine on Cardio-Cerebrovascular Disease

基　　金：福建省自然科学基金项目(青年创新)(No.2020J05283);中国人民解放军联勤保障部队第九OO医院院内课题杰青项目(No.2021JQ12)。

摘　　要：目的:探究微血管内皮细胞来源的外泌体对血管性痴呆(VD)模型小鼠脑血管形及神经损伤修复的影响。方法:采用超高速离心法提取小鼠脑微血管内皮细胞bEnd.3的外泌体,透射电子显微镜(TEM)观察外泌体形态,纳米颗粒追踪分析(NTA)测定外泌体径粒分布情况,蛋白质免疫印迹法(Western Blot)测定外泌体标志蛋白CD 63、TSG101、Alix表达;将40只小鼠按照随机数字表法分为假手术(Sham)组、模型(Model)组、外泌体(b-Exo)组、阳性对照(DP)组,每组10只,除假手术组,其余3组小鼠均采用双侧颈总动脉缩窄法构建VD模型,b-Exo组腹腔注射100μL的b-Exo(100μg/mL),DP组腹腔注射盐酸多奈哌齐(1 mg/kg),假手术组和模型组腹腔注射等体积生理盐水,每日1次,持续28 d;Morris水迷宫行为学实验分析小鼠学习记忆能力,苏木精-伊红(HE)染色法观察小鼠海马组织病理学变化,免疫荧光染色检测小鼠脑组织血管形成情况,Western Blot检测小鼠海马组织中血管内皮生长因子(VEGF)蛋白表达水平,免疫荧光染色检测小鼠海马组织中神经元特异核蛋白(NeuN)表达,酶联免疫吸附法(ELISA)测定小鼠血清脑损伤相关因子星形胶质源性蛋白(S-100β)与神经元特异性烯醇化酶(NSE)水平。结果:从bEnd.3细胞分离的颗粒物呈圆形囊泡状,CD 63、TSG101及Alix蛋白均高表达,说明该颗粒物为外泌体,记为b-Exo;与假手术组比较,模型组小鼠逃避潜伏期延长,通过目标象限次数和在目标象限停留时间百分比减少,海马CA1区神经元细胞数量减少,出现明显病理学损伤,血管形成减少,VEGF蛋白相对表达量下调,NeuN表达减少,血清S-100β与NSE水平升高,差异均有统计学意义(P<0.05);与模型组比较,b-Exo组和DP组小鼠逃避潜伏期缩短,通过目标象限次数和在目标象限停留时间百分比增加,海马CA1区损伤明显减轻,神经元细胞数量有所增加,血管形成增加,VEGF蛋白相对表达量上调(P<0.05Objective:To investigate the effects of exosomes derived from microvascular endothelial cells on cerebrovascular formation and nerve injury repair in vascular dementia(VD)model mice.Methods:The exosomes of mouse brain microvascular endothelial cells bEnd.3 were extracted by ultra-high-speed centrifugation method.The morphology of exosomes was observed by transmission electron microscopy(TEM),and the diameter distribution of exosomes was detected by nanoparticle tracking analysis(NTA).The expressions of exosomal marker proteins CD 63,TSG101 and Alix were detected by Western Blot.Forty mice were divided into Sham group,Model group,exosome group(b-Exo group)and positive control group(DP group)according to numerical random table method,with 10 mice in each group.Except for sham group,the VD model of the other 3 groups was constructed by bilateral common carotid artery constriction method,b-Exo group was intraperitoneally injected with 100μL b-Exo(100μg/mL),DP group was intraperitoneally injected with Donepezil hydrochloride(1 mg/kg),sham group and model group were intraperitoneally injected with equal volume normal saline,once a day,for 28 days.Morris water maze behavior experiment was used to analyze the learning and memory ability of mice,hematoxylin-eosin(HE)staining was used to observe the histopathological changes of mice′s hippocampus,immunofluorescence staining was used to detect the formation of blood vessels in mouse brain tissue,and Western Blot was used to detect the expression level of vascular endothelial growth factor(VEGF)protein in mouse hippocampus.The expression of neuron-specific nucleoprotein(NeuN)in the hippocampus of mice was detected by immunofluorescence staining,and the levels of astroglia-derived protein(S-100β)and neuron-specific enolase(NSE)in serum of mice were detected by enzyme-linked immunosorbent assay(ELISA).Results:The particles isolated from bEnd.3 cells showed a round vesicular shape,and CD 63,TSG101 and Alix proteins were highly expressed,indicating that the particles were e

关 键 词：血管性痴呆 微血管内皮细胞 外泌体 血管形成 神经保护 实验研究 

分 类 号：R749.13[医药卫生—神经病学与精神病学] R-332[医药卫生—临床医学]