外泌体介导miR-519c-5p增强脂多糖诱导的血管内皮细胞凋亡作用  被引量:4

Enhancement of lipopolysaccharide-induced apoptosis of vascular endothelial cellsvia exosome-mediated miRNA-519c-5p

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作  者:黄发 吕嘉贤 许静红[1,2] 姚纪友 童荔 胡晓光[1] 朱艳平[1] 曹璐[1] 蔡常洁[1] HUANG Fa;Lü Jiaxian;XU Jinghong;YAO Jiyou;TONG Li;HU Xiaoguang;ZHU Yanping;CAO Lu;CAI Changjie(The First Affiliated Hospital of Sun YatSen University,Guangzhou 510080,China;不详)

机构地区:[1]中山大学附属第一医院,广州510080 [2]广东省器官捐献与移植免疫重点实验室,广东省器官移植科技合作基地,广州510080 [3]广州市第一人民医院,广州510000

出  处:《实用医学杂志》2020年第6期722-728,共7页The Journal of Practical Medicine

基  金:广州市科技计划项目(编号:201704020153);广东省器官捐献与移植免疫重点实验室建设项目(编号:2013A061401007);广东省器官移植国际合作基地建设项目(编号:2015B050501002)。

摘  要:目的探讨外泌体介导的miR-519c-5p是否能增强脂多糖诱导的血管内皮细胞凋亡作用。方法收集大量人脐带静脉内皮细胞(HUVECs)上清液,通过超速离心法制备大量外泌体(exosomes),制备大量无任何处理的blank-exosomes和过表达miR-519c-5p的miR-519c-5p-exosomes;同时予以LPS 10μg/mL刺激HUVECs 6 h构建脓毒症模型,再与分别与上述两种外泌体共培养12 h。实验设立3个组:HUVECs+blank-exosomes为空白组,HUVECs+LPS+blank-exosomes、HUVECs+miR-519c-5p-exosomes为对照组,HUVECs+LPS+miR-519c-5p-exosomes为实验组。通过qRT-PCR法检测miR-519c-5p在外泌体及HUVECs中的表达,通过CCK-8法与流式细胞术分别检测各组内皮细胞增殖和凋亡的情况。结果经qRT-PCR验证转染的过表达exosomes较blank-exosomes与mimic NC-exosomes miR-519c-5p表达升高约13倍(P<0.001)。同时,CCK-8测得实验组OD值均低于空白组和对照组(0.458 vs.1.388,0.458 vs.0.843,P<0.05),流式细胞术测得实验组细胞凋亡比例均高于空白组和对照组(7.197%vs.0.647%,P<0.001;7.197%vs.3.423%,P=0.001)。结论外泌体介导的miR-519c-5p能增强脂多糖诱导的血管内皮细胞凋亡作用。Objective To identify whether the exosomal mediated delivery of functionally active miRNA-519 c-5 p could enhance apoptosis of LPS-induced vascularendothelial cells.Methods Exosomes were collected from Human umbilical veinendothelial cells(HUVECs)supernatants by ultracentrifugation.Some exosomes transfected with miR-519 c-5 p mimic were for miR-519 c-5 p-exosomes,and others untreated exosomes for blank exosomes.LPS was also applied 10μg/ml to stimulate HUVECs for 6 h to construct a sepsis model,and then was co-cultured with exosomes for 12 h.HUVECs+blank-exosomes was set as blank group;HUVECs+LPS+blank-exosomes,HUVECs+miR-519 c-5 p-exosomes as control group,and HUVECs+LPS+miR-519 c-5 p-exosomes as experimental group.qRT-PCR was performed to detect the expression of miR-519 c-5 p in exosomes and HUVECs.The effects of endothelial cell proliferation and apoptosis were detected by CCK-8 and flow cytometry,respectively.Results The expression of overexpressing exosomes was significantly increased by 13-fold compared with blank-exosomes and mimic NC-exosomes miR-519 c-5 p by qRT-PCR(P<0.001).At the same time,the OD value of the experimental group was lower than that of the blank group and the control group(0.458 vs.1.388,0.458 vs.0.843,P<0.05).The proportion of apoptotic cells of the experimental group was higher in the blank group and the control group(7.197%vs.0.647%,P<0.001;7.197%vs.3.423%,P=0.001).Conclusion Exosome-mediated miR-519 c-5 p can enhance lipopolysaccharide-induced apoptosis of vascular endothelial cells.

关 键 词:外泌体 脓毒症 凋亡 人脐带静脉内皮细胞 MIRNA 

分 类 号:R319[医药卫生—基础医学]

 

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