摘要
目的:探讨携带血小板反应素-1(thrombospondin-1,TSP-1)的人脐带间充质干细胞(human umbilical cord mesenchymal stem cell,hUCMSC)来源外泌体对视网膜静脉阻塞(retinal vein occlusion,RVO)模型大鼠的保护作用。方法:将hUCMSC随机分为hUCMSC组、NC-hUCMSC组、TSP-1-hUCMSC组,NC-hUCMSC组和TSP-1-hUCMSC组分别利用含TSP-1基因序列慢病毒、阴性对照慢病毒感染hUCMSC,并通过荧光倒置显微镜、实时荧光定量PCR和Western blot测定细胞转染效果;分离携带TSP-1基因的hUCMSC来源的外泌体,透射电镜观察形态,纳米粒子跟踪分析法测定直径,Western blot检测外泌体标志蛋白CD9、CD63及Alix的表达;采用激光光动力法建立SD大鼠RVO模型,并分为模型组、hUCMSC-Exo组、TSP-1-hUCMSC-Exo组,每组10只,另取10只健康大鼠作为对照组,给予hUCMSC-Exo组结膜下注射100μL hUCMSC来源外泌体,TSP-1-hUCMSC-Exo组注射100μL含TSP-1慢病毒感染的hUCMSC来源外泌体。RVO模型建立后1d、7d、14d、21d,观察眼底图像;21 d后,眼底血管荧光造影(fluorescein fundus angiography,FFA)检查血管病变,HE染色观察视网膜组织病理学变化,实时荧光定量PCR和Western blot检测视网膜组织内缺氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)、血管内皮生长因子(vascular endothelial growth factor,VEGF)和血管细胞间黏附分子-1(vascular cell adhesion molecule-1,VCAM-1)在mRNA和蛋白上的表达水平。结果:倒置荧光显微镜可见慢病毒转染的细胞内有明显的绿色荧光蛋白表达,且TSP-1-hUCMSC组的hUCMSC中TSP-1 mRNA和蛋白相对表达量均显著升高(P<0.05),表示细胞转染成功;分离的颗粒物为球状,直径大约分布在100 nm,CD9、CD63及Alix蛋白均高表达,说明成功分离到外泌体;与对照组比较,模型组大鼠眼内在造模1、7、14和21d均出现不同程度的白色闭塞血管及斑块,判定造模成功;此外,模型组视网膜血管可见静脉迂曲扩张,粗细不均,血管壁荧光着染及�
Objective:To investigate the protective effect of human umbilical cord mesenchymal stem cell(hUCMSC)-derived exosomes carrying thrombospondin-1(TSP-1)in a rat model of retinal vein occlusion(RVO).Methods:The hUCMSCs were randomly divided into hUCMSC group,NC-hUCMSC group and TSP-1-hUCMSC group,the NC-hUCMSC group and TSP-1-hUCMSC group were infected with the lentivirus containing TSP-1 gene sequence and the negative control lentivirus,respectively,and the cell transfection effect was determined by fluorescent inverted microscope,real-time fluorescent quantitative PCR and Western blot.The hUCMSC-derived exosomes carrying the TSP-1 gene were isolated,the morphology was observed by transmission electron microscopy,the diameter was determined by the nanoparticle tracking analysis method,and the expression of the exosomal marker proteins CD9,CD63 and Alix was detected by Western blot.The RVO model of SD rats was established by laser photodynamic method and divided into model group,hUCMSC-Exo group,TSP-1-hUCMSC-Exo group,each with 10 rats,and another 10 healthy ratswerewolf made the control group.The hUCMSC-Exo group was subconjunctivally injected with 100μl of hUCMSC-derived exosomes,and the TSP-1-hUCMSC-Exo group was injected with 100μL of hUCMSC-derived exosomes containing TSP-1 lentivirus infection.1 day,7 days,14 days,21 days after the establishment of the RVO model,the fundus images were observed.21 d later,the vascular lesions were examined by fluorescein fundus angiography(FFA),and the histopathological changes of the retina were observed by HE staining,Real-time fluorescent quantitative PCR and Western blot were used to detect the mRNA and protein levels of hypoxia-inducible factor-1α(HIF-1α),vascular endothelial growth factor(VEGF)and vascular cell adhesion molecule-1(VCAM-1)in retinal tissues.Results:The inverted fluorescence microscope showed that there was obvious green fluorescent protein expression in the cells transfected by the lentivirus,and the relative expression of TSP-1 mRNA and protein in hUC
作者
李霞
张茂菊
LI Xia;ZHANG Maoju(Enshi Central Hospital,Hubei Enshi 445000,China)
出处
《河北医学》
CAS
2022年第7期1080-1087,共8页
Hebei Medicine
基金
湖北省恩施州医疗卫生类指导性项目,(编号:JCY2019000006)。
