摘要
目的探讨黄芪甲苷(AstragalosideⅣ,AS-Ⅳ)干预的人内皮祖细胞外泌体(Endothelial progenitor cells derived exosomes,EPC-Exos)对高糖诱导损伤人脐血间充质干细胞(Human umbilical cord blood mesenchymal stem cells,hUCBMSCs)向内皮分化的影响。方法体外分离和培养人内皮祖细胞(Endothelial Progenitor Cells,EPCs),同时体外分离和培养hUCBMSCs,取P4代细胞分别进行成骨、成软骨和成脂诱导分化鉴定。将鉴定成功的EPCs分别用100mg·L-1的黄芪甲苷和等量的PBS干预,培养24 h后收集两组细胞上清液中外泌体。采用透射电子显微镜观察EPC-Exos的形态,利用纳米粒子追踪分析(Nanoparticle Tracking Analysis,NTA)技术检测EPC-Exos的粒径,Western blot技术进行外泌体特征性标志物CD9、CD63和TSG101的检测。将hUCBMSCs用30 mmol·L-1的葡萄糖预处理120h后,随机分为实验组和对照组,同时设置正常组。三组细胞培养24 h后,通过Matrigel体外成管实验研究EPC-Exos对hUCBMSCs成管分化的影响。免疫荧光检测hUCBMSCs表达CD31、vWF等内皮细胞特异标志物的情况。结果光镜下hUCBMSCs边缘清楚,形态均一,排列呈漩涡状,3系细胞分化为典型图像。透射电镜观察分离及提纯后黄芪甲苷干预EPC-Exos为包膜完整的圆形或椭圆形微囊泡结构;NTA技术检测97.6%人EPC-Exos为直径在81.4-142.1nm之间的微囊泡;EPC-Exos表面特异标志物CD9、CD63、TSG101呈阳性,以上实验证实成功提取EPC-Exos。Matrigel成管实验显示,与对照组相比,实验组MSCs细胞体外成管能力显著增强,差异显著(P<0.001)。免疫荧光检测显示,与对照组相比,实验组MSCs表达内皮细胞特异性标志物CD31、vWF能力显著增强,差异显著(P均<0.01)。结论黄芪甲苷干预的EPC-Exos可有效恢复高糖受损人间充质干细胞的成管功能且显著改善其向内皮分化能力。
Objective To investigate the effect of AstragalosideⅣ(AS-Ⅳ)interfered exosomes(EPC-exos)on endothelial differentiation of human umbilical cord blood mesenchymal stem cells(hUCBMSCs)induced by high glucose.Methods Human Endothelial Progenitor Cells(EPCs)were isolated and cultured in vitro.At the same time,hUCBMSCs were isolated and cultured in vitro,and the P4 generation cells were selected for osteogenic,chondrogenic and adipogenic differentiation identification respectively.The identified EPCs were treated with 100 mg·L-1AstragalosideⅣand the same amount of PBS respectively.Exosomes in the supernatant of cells of the two groups were collected after 24 h.The morphology of EPC-Exos was observed by transmission electron microscope.The particle size of EPC-Exos was measured by Nanoparticle Tracking Analysis(NTA)technology.Characteristic markers of exosomes,CD9,CD63 and TSG101,were detected by Western blot.The hUCBMSCs were pretreated with 30 mmol·L-1glucose for 120 h,and then randomly divided into experimental group and control group,while the normal group was set.After cultured for 24 h,the effect of EPC-Exos on the differentiation of MSCs was studied by matrigel tube formation assay.Expression of CD31 and VWF which are endothelial cell specific markers in MSCs was detected by immunofluorescence.Results Under light microscope,the edge of hUCBMSCs was clear.The shape was uniform.The arrangement was swirling,and the 3-line cells differentiated into typical images.Transmission electron microscopy observation showed that after separation and purification.Astragaloside IV interfered EPC-Exos were round or elliptical vesicles with complete envelopments;NTA technology detected 97.6%of EPC-Exos as round vesicles with diameter between 81.4 nm and 142.1 nm;CD9,CD63 and TSG101,the specific markers on the surface of EPC-Exos were positive.The above experiments confirmed the successful extraction of EPC-Exos.Matrigel tube formation assay showed that compared with the control group,the tubular-forming ability of MSCs cel
作者
彭阿建
欧阳范馨
张熙
谭梅鑫
梁文菲
朱晨鸿
刘锦清
张璐瑶
肖郁婷
熊武
Peng Ajian;Ouyang Fanxin;Zhang Xi;Tan Meixin;Liang Wenfei;Zhu Chenhong;Liu Jinqing;Zhang Luyao;Xiao Yuting;Xiong Wu(College of Integrated Traditional Chinese and Western Medicine,Hunan University of Chinese Medicine,Changsha 410208,China;The First Affiliated Hospital of Hunan University of Chinese Medicine,Changsha 410007,China;Hunan Brain Hospital,Clinical Medical School of Hunan University of Chinese Medicine,Changsha 410007,China)
出处
《世界科学技术-中医药现代化》
CSCD
北大核心
2022年第4期1593-1602,共10页
Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology
基金
国家自然科学基金委员会青年科学基金项目(81904217)
基于外泌体miR-126调控PIK3R2/SPRED1探讨黄芪甲苷促糖尿病皮肤溃疡,负责人:熊武
国家级大学生创新创业训练计划项目(S202010541015)
从外泌体miR-146a-5p介导TRAF6/NF-κB信号通路探讨黄芪甲苷调控高糖受损内皮细胞炎症反应的机制研究,负责人:彭阿建
湖南省教育厅科学研究立项优秀青年项目(20B437)
从外泌体miR-21调控细胞自噬水平探讨黄芪甲苷保护高糖受损内皮细胞的机制研究,负责人:熊武。
关键词
黄芪甲苷
内皮祖细胞
外泌体
间充质干细胞
内皮分化
高糖环境
AstragalosideⅣ
Endothelial progenitor cells
Exosomes
Mesenchymal stem cells
Endothelial differentiation
High glucose environment
