间充质干细胞来源的外泌体调控miR-143对大鼠颅内动脉瘤形成的影响机制  

The effect of mesenchymal stem cells-derived exosomes on the formation of intracranial aneurysm in rats by regulating miR143

作　　者：冯利飞[1] 杨歌 梁超[1] 陈芳鑫 FENG Lifei;YANG Ge;LIANG Chao;CHEN Fangxin(Department of Neurosurgical,Xingtai People's Hospital,Xingtai 054000,Hebei,China;Department of Anesthe-siology,Hebei General Hospital,ShiJiazhuang 050000,Hebei,China;Department of Hospital Infection,Xingtai People's Hospital,Xingtai 054000,Hebei,China)

机构地区：[1]邢台市人民医院神经外二科,邢台054000 [2]邢台市人民医院感染管理科,邢台054000 [3]河北省人民医院麻醉科,石家庄050000

出　　处：《医学研究与战创伤救治》2024年第6期575-587,共13页Journal of Medical Research & Combat Trauma Care

摘　　要：目的 研究间充质干细胞(MSCs)来源的外泌体调控miR-143对大鼠颅内动脉瘤(IA)形成的影响及机制。方法 原代分离大鼠骨髓MSCs(BMSCs)和脑血管平滑肌细胞(VSMCs)。将VSMCs分为PBS组、外泌体-miR-NC组(ExosomemiR-NC)、Exosome-miR-143组、Exosome-miR-143+GW4869组、过表达对照组(oeNC)、过表达KLF5组(oeKLF5)和oeKLF5+Exosome-miR-143组。超速离心法分离BMSCs来源的外泌体,并用透射电镜和纳米示踪分析鉴定外泌体。PKH26染色检测外泌体转移。miR-143 mimic和miR-NC转染BMSCs或VSMCs。实时荧光定量PCR检测miR-143和KLF5 mRNA的表达,Western blot检测相关蛋白的表达。CCK-8实验、EdU实验、流式细胞术实验、划痕实验和Transwell迁移实验检测VSMCs的活力、增殖、凋亡和迁移能力。双荧光素酶报告基因系统分析miR-143与KLF5的关系。构建大鼠IA模型,研究BMSCs来源的外泌体miR-143在体内对IA形成的影响。结果 与细胞裂解液组相比,miR-143在BMSCs外泌体中表达显著增加(P<0.01),且可通过外泌体转移到VSMCs中。与Exosome-miR-NC组相比,Exosome-miR-143组VSMCs的活力增加(P<0.01),而EdU阳性细胞数、S期细胞数、凋亡和迁移数显著减少(P<0.01);与Exosome-miR-143组相比,Exosome-miR-143+GW4869组细胞活力降低(P<0.01),而EdU阳性细胞数、S期细胞数、凋亡和迁移数明显增加(P<0.01)。与miR-NC或Control组相比,miR-143、Exosome-miR-143和ExosomemiR-NC组WT-KLF5的荧光素酶报告基因活性及KLF5的mRNA和蛋白表达水平降低(P<0.01),而MUT-KLF5的荧光素酶报告基因活性不变(P>0.05)。与oeNC组相比,oeKLF5组细胞活力显著降低(P<0.01),EdU阳性细胞数、S期细胞数、凋亡和迁移数明显增加(P<0.01);与oeKLF5组相比,oeKLF5+Exosome-miR-143组细胞活力明显增加(P<0.01),而EdU阳性细胞数、S期细胞数、凋亡和迁移数明显减少(P<0.01)。体内实验结果与细胞实验相一致。结论 BMSCs来源的外泌体miR-143通过靶向KLF5抑制大鼠IA的形�Objective This study aims to explore the effect and mechanism of mesenchymal stem cells(MSCs)-derived exosomes regulating miR-143 on the formation of intracranial aneurysm(IA).Methods Primary rat bone marrow mesenchymal stem cells(BM-SCs)and brain vascular smooth muscle cells(VSMCs)were isolated.VSMCs were divided into PBS Group,Exosome-miR-NC Group,Exo-some-miR-143 Group,Exosome-miR-143+GW4869 Group,Overex-pression Control Group(oeNC Group),KLF5-overexpression Group(oeKLF5 Group)and oeKLF5+Exosome-miR-143 Group.BMSCs-derived exosomes were isolated by ultracentrifugation and identified by transmission electron microscopy and nano-tracer analysis.PKH26 staining was used to detect exosome transfer.Transfection of miR-143 mimic and miR-NC to BMSCs and VSMCs was performed.qRT-PCR was performed to determine miR-143 expression.West-ern blot was used to detect protein expressions.CCK8,EdU,flow cytometry,wound healing and Transwell assays were conducted to measure the viability,proliferation,apoptosis,and migration abilities of VSMCs.The dual luciferase reporter gene system was used to analyze the relationship between miR-143 and KLF5.A rat IA model was established to study the effects of BMSCs-derived exosome-miR-143 on the formation of IA in vivo.Results Compared with cell lysate Group,miR-143 expression in BMSCs exosomes was sig-nificantly increased(P<0.01),and it could be transferred into VSMCs through exosomes.Compared with the exosome-miR-NC Group,the viability of VSMCs in the Exosome-miR-143 Group was increased(P<0.01),while the number of EdU-positive cells,S-phase cells,apoptotic and migratory cells were significantly decreased(P<0.01).Compared with the exosome-miR-143 group,the cell viability in exosome-miR-143+GW4869 group was decreased(P<0.01),while the number of EdU-positive cells,S-phase cells,apoptotic and mi-gratory cells were significantly increased(P<0.01).Compared with the miR-NC or the control group,the luciferase reporter gene activi-ty of WT-KLF5 and the mRNA and protein levels of KLF5 in the miR-143

关 键 词：间充质干细胞 外泌体 MIR-143 KLF5 颅内动脉瘤 

分 类 号：R739.41[医药卫生—肿瘤]