内皮微粒对血管内皮细胞衰老的影响及机制研究  

Effect of endothelial microparticles on vascular endothelial cell senescence

作　　者：于博文 王雪[1] 胡艳红 杨静[1] 李雪丽[1] 修成奎[1] 雷燕[1] YU Bo-wen;WANG Xue;HU Yan-hong;YANG Jing;LI Xue-li;XIU Chengkui;LEI Yan(Beijing Key Laboratory of Research of Chinese Medicine on Prevention and Treatment for Major Diseases,Medical Experi-ment Center,China Academy of Chinese Medical Sciences,Beijing 100700,China;Center for Post-doctoral Studies,Chi-na Academy of Chinese Medical Sciences,Beijing 100700,China)

机构地区：[1]中国中医科学院医学实验中心,北京市中医药防治重大疾病基础研究重点实验室,北京100700 [2]中国中医科学院博士后科研流动站,北京100700

出　　处：《中国病理生理杂志》2021年第5期825-833,共9页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81673822);中国中医科学院院级自主选题(No.ZZ2019014)。

摘　　要：目的:探讨内皮微粒(EMPs)对血管内皮细胞衰老的影响及作用机制。方法:通过体外传代,将人脐静脉内皮细胞(HUVECs)培养至第10~12代,建立复制性衰老模型。实验分为年轻组(2~4代)、衰老组(10~12代)及EMPs干预组(提取衰老细胞产生的EMPs来干预年轻细胞)。结合衰老相关β-半乳糖苷酶(SA-β-gal)染色法和PI单染法检测细胞周期两种方法判断细胞衰老情况。采用两步离心法分离出EMPs;采用醋酸双氧铀负染法于透射电镜下观察EMPs的大小及形态;流式细胞术检测CD31^(+)或Annexin V^(+)的EMPs;DCFDA染色检测细胞内活性氧簇(ROS)的表达。结果:与年轻组相比,衰老组细胞SA-β-gal活性显著增强(P<0.01),细胞周期阻滞于S期(P<0.01),并且细胞内ROS水平显著升高(P<0.01);衰老组细胞分泌CD31^(+)EMPs及Annexin V^(+)EMPs的数量均显著高于年轻组(P<0.05)。电镜下EMPs直径在0.1~1μm之间,形态呈圆形或椭圆形,其囊泡膜结构完整,边缘清楚,轮廓清晰。与年轻组相比,EMPs干预组细胞SA-β-gal活性增强(P<0.01),细胞周期阻滞于S期(P<0.01),细胞内ROS水平显著升高(P<0.01),并且增高的细胞内ROS可部分被apocynin(NADPH氧化酶抑制剂)或rotenone(线粒体氧化酶抑制剂)抑制(P<0.05)。结论:衰老HUVECs分泌的EMPs多于年轻HUVECs;衰老HUVECs分泌的EMPs可导致年轻HUVECs早衰,其机制可能与EMPs通过激活NADPH氧化酶及线粒体氧化酶,进而诱导细胞内ROS水平增高有关。AIM:To investigate the effect of endothelial microparticles(EMPs)on vascular endothelial cell senescence.METHODS:Human umbilical vein endothelial cells(HUVECs)were cultured to passage 10-12 in vitro to establish a replicative senescence model.The experiment was divided into young group(passage 2-4),aged group(pas-sage 10-12)and EMPs intervention group.Senescence-associatedβ-galactosidase(SA-β-gal)staining and cell cycle PI staining were used to determine cell senescence.EMPs were extracted by two-step centrifugation.The size and morpholo-gy of EMPs were observed under transmission electron microscope with uranium dioxide acetate negative staining.Amount of CD31^(+)EMPs and Annexin V^(+)EMPs were detected by flow cytometry.Intracellular reactive oxygen species(ROS)were detected by DCFDA staining.RESULTS:Compared with young group,aged group was characterized by higher SA-β-gal activity and higher ROS levels(P<0.01),the cell cycle arrested in S phase(P<0.01).The number of CD31^(+)EMPs and Annexin V^(+)EMPs secreted by aged HUVECs were significantly higher than those in young group(P<0.05).The diameter of EMPs ranged from 0.1 to 1μm.The shape of the EMPs was round or oval,with complete vesicle membrane structure,clear edge and clear outline.Furthermore,the results in EMPs intervention group showed increased SA-β-gal activity(P<0.01),induction of S phase arrest and increased intracellular ROS level(P<0.01).The increased intracellular ROS was partially inhibited by apocynin or rotenone(P<0.05).CONCLUSION:EMPs secreted by aged HUVECs are more than those secreted by young HUVECs.EMPs secreted by aged HUVECs leads to premature senescence of young HUVECs,and the mechanism may be related to increase intracellular ROS level by activating NADPH oxidase and mitochondrial oxi-dase.

关 键 词：内皮微粒 细胞衰老 活性氧 人脐静脉内皮细胞 

分 类 号：R329.25[医药卫生—人体解剖和组织胚胎学] R363.2[医药卫生—基础医学]