解剖学报 ›› 2024, Vol. 55 ›› Issue (6): 761-768.doi: 10.16098/j.issn.0529-1356.2024.06.015

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外泌体传递微小RNA-145对冠状动脉粥样硬化性心脏病大鼠血小板活化及血管内皮功能的影响
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王建美 金卫东 刘振 刘烝昊*    

  1. 新乡市中心医院心血管内科,河南 新乡 453000
  • 收稿日期:2023-07-06 修回日期:2023-09-03 出版日期:2024-12-06 发布日期:2024-12-06
  • 通讯作者: 刘烝昊 E-mail:aphroditelove@163.com
  • 基金资助:
    河南省医科技攻关计划项目

Effect of microRNA-145 delivered by exosomes on platelet activation and vascular endothelial function in rats with coronary atherosclerotic heart disease

WANG  Jian-mei JIN  Wei-dong  LIU  Zhen  LIU  Zheng-hao*   

  1. Department of Cardiology, Xinxiang Central Hospital, He’nan Xinxiang 453000, China
  • Received:2023-07-06 Revised:2023-09-03 Online:2024-12-06 Published:2024-12-06
  • Contact: LIU Zheng-hao E-mail:aphroditelove@163.com

摘要:

目的 探讨外泌体(Exo)传递微小RNA(miR-145对冠状动脉粥样硬化性心脏病(CAHD,简称冠心病)模型大鼠血小板活化以及血管内皮功能的影响及分析。 方法  通过Real-time PCR检测miR-阴性对照(NC)和miR-145转染HEK239细胞的效果,并转染从转染miRNA-NC、miRNA-145的HEK239细胞中分离的Exo,透射电子显微镜下观察Exo形态并分析其径粒分布情况,Western blotting测定CD81、热休克蛋白70(HSP70)及肿瘤易感基因101(TSG101)蛋白表达;实验分为对照组、模型组、miR-NC Exo组、miR-145 Exo组,每组8只大鼠,处理结束后,动物超声诊断仪测定大鼠射血分数(EF)、短轴缩短率(FS)、左心室舒张末期内径(LVIDD)、左心室收缩末期内径(LVIDS),HE染色观察大鼠心肌组织与主动脉组织的病理改变,ELISA测定大鼠血清血小板活化因子(PAF)、β血小板球蛋白(β-TG)、血小板膜糖蛋白Ⅱa/Ⅲb(GPⅡa/Ⅲb)、一氧化氮(NO)、内皮素-1(ET-1)和血管内皮生长因子(VEGF)的含量,免疫组织化学染色检测大鼠主动脉组织中内皮型一氧化氮合酶(eNOS)的表达。 结果  转染miR-145的HEK239细胞中miR-145相对表达量显著高于未转染及转染miR-NC的HEK239细胞(P <0.05),分离的颗粒物呈典型的杯状或盘状囊泡,直径大多分布在100 nm,CD81、HSP70及TSG101蛋白均高表达,且转染miR-NC的Exo中miR-145相对表达量显著低于转染miR-145的 Exo(P <0.05)。与miR-NC Exo组比较,miR-145 Exo组大鼠EF、FS显著升高(P <0.05),LVIDD、LVIDS显著减小(P <0.05),心肌组织与主动脉组织病理变化改善效果更好,血清中PAF、β-TG、GPⅡa/Ⅲb、ET-1和VEGF含量显著降低(P <0.05),NO含量也显著升高(P <0.05),主动脉组织eNOS 阳性表达率也显著增加(P <0.05)。 结论 Exo传递miR-145能够抑制冠心病模型大鼠血小板活化,改善血管内皮功能,对冠心病模型大鼠起到保护作用。 

关键词: 微小RNA-145, 血小板活化, 外泌体, 血管内皮功能, 冠状动脉粥样硬化性心脏病, 实时定量聚合酶链反应, 免疫印迹法, 大鼠 

Abstract:

 Objective To investigate the effects of microRNA (miR)-145 delivered by exosomes (Exo) on platelet activation and vascular endothelial function in rats with coronary atherosclerotic heart disease (CAHD).  Methods   HEK239 cells were transfected with miR-negative control(NC) and miR-145, and the transfection effect was detected by Realtime PCR. Exo was isolated from HEK239 cells transfected with miRNA-NC and miR-145. The morphology and size distribution were observed by transmission electron microscopy. The expressions of CD81, heat shock protein 70(HSP70)and tumor susceptibility gene 101(TSG101)were detected by Western blotting. The experiment included control group, model group, miR-NC Exo group and miR-145 Exo group, with 8 rats in each group. After treatment, the ejection fraction (EF), fractional shortening rate (FS), left ventricular enddiastolic diameter (LVIDD) and left ventricular end-systolic diameter (LVIDS) of rats were determined by ultrasonic diagnostic instrument. HE staining was performed to observe the pathological changes of myocardial tissue and aortic tissue, and ELISA was used to determine serum platelet activating factor (PAF), β-platelet globulin (β-TG), platelet membrane glycoprotein Ⅱa/Ⅲb (GPⅡa/Ⅲb), nitric oxide (NO), endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF). Immunohistochemical staining was used to detect the expression of endothelial nitric oxide synthase (eNOS) in aorta tissue.  Results  The relative expression level of miR-145 in HEK239 cells transfected with miR-145 was significantly higher than that in untransfected and transfected miR-NC cells (P <0.05). The isolated particles showed typical cup-shaped or disk-shaped vesicles, most of which were distributed at 100 nm in diameter. CD81, HSP70 and TSG101 proteins were highly expressed, and the relative expression level of miR-145 in Exo transfected with miR-NC was significantly lower than that in Exo transfected with miR-145 (P <0.05). Compared with the miR-NC Exo group, EF and FS of miR-145 Exo group increased significantly(P <0.05), while LVIDD and LVIDS decreased significantly(P <0.05), and the pathological changes of myocardial tissue and aortic tissue were better improved. The contents of PAF, β-TG, GPⅡa/Ⅲb, ET-1 and VEGF in serum were further significantly decreased (P <0.05), while the content of NO was also significantly increased (P <0.05), and the positive expression rate of eNOS in aortic tissue was further significantly increased (P <0.05).  Conclusion  MiR-145 delivered by Exo could inhibit platelet activation and improve vascular endothelial function in coronary heart disease model rats, and plays a protective role in coronary heart disease model rats. 

Key words: MicroRNA-145, Platelet activation, Exosome, Vascular endothelial function, Coronary atherosclerotic heart disease, Real-time PCR, Western blotting, Rat

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