血栓闭塞性脉管炎患者血浆源性外泌体miR-223-5p对人血管平滑肌细胞的影响  

Effect of plasma derived exosomal miR-223-5p on human vascular smooth muscle cells in patients with thromboangiitis obliterans

作　　者：陈波 林学广 邓颖 王博 童进东 余波 史卫军 汤敬东 CHEN Bo;LIN Xue-guang;DENG Ying;WANG Bo;TONG Jin-dong;YU Bo;SHI Wei-jun;TANG Jing-dong(Department of Vascular Surgery,Shanghai Pudong Hospital-Fudan University Pudong Medical Center,Shanghai 201399,China;Shanghai Key Laboratory of Vascular Lesions Regulation and Remodeling,Shanghai 201399,China;Department of Vascular Surgery,Huashan Hospital,Fudan University,Shanghai 200040,China)

机构地区：[1]上海市浦东医院-复旦大学附属浦东医院血管外科,上海201399 [2]上海市血管病变调控与重塑重点实验室,上海201399 [3]复旦大学附属华山医院血管外科,上海200040

出　　处：《复旦学报（医学版）》2022年第5期677-689,共13页Fudan University Journal of Medical Sciences

基　　金：上海市浦东新区卫健委卫生计生科研项目(PW2019E-5);上海市卫健委临床研究项目(202150004)。

摘　　要：目的探究血栓闭塞性脉管炎(thromboangiitis obliterans,TAO)患者血浆源性外泌体中miRNAs对人血管平滑肌细胞(human vascular smooth muscle cell,HVSMC)的影响。方法从TAO患者和健康对照的血浆中提纯外泌体,进行miRNA测序。通过生物信息学分析鉴定差异表达的miRNA(differentially expressed miRNA,DEmiRNA),用RT-qPCR进一步验证。将PKH67荧光标记的外泌体与HVSMC共培养,采用CCK-8法和流式细胞术分别检测HVSMC的细胞活力和凋亡。使用双荧光素酶报告分析并确认miR-223-5p的下游靶点。结果在TAO患者和健康对照之间共发现39个DE-miRNA,其中miR-223-5p是显著上调的miRNA。TAO患者血浆外泌体或miR-223-5p模拟物能降低HVSMC的细胞活力并促进细胞凋亡,miR-223-5p抑制剂可削弱TAO患者血浆源性外泌体的抑制和促凋亡作用。血管内皮细胞黏附分子1(vascular endothelial cell adhesion molecule-1,VCAM-1)和胰岛素样生长因子1受体(insulin-like growth factor-1 receptor,IGF-1R)的表达被TAO患者血浆源性外泌体和miR-223-5p模拟物下调,这一下调作用可被miR-223-5p抑制剂所抑制。双荧光素酶报告提示VCAM-1是miR-223-5p的靶点。结论TAO患者血浆源性外泌体可能通过miR-223-5p/VCAM-1途径抑制HVSMC的细胞活力并促进细胞凋亡,从而在TAO的发生发展中起作用。Objective To investigate the effect of miRNAs in plasma exosomes of patients with thromboangiitis obliterans(TAO)on human vascular smooth muscle cells(HVSMCs).Methods Exosomes were purified from the plasma of TAO patients and healthy controls,and miRNA was sequenced.The differentially expressed miRNA(DE-miRNA)were identified by bioinformatics analysis and further verified by RT-qPCR.Then,PKH67 fluorescently labeled exosomes were co-cultured with HVSMCs.Cell viability and apoptosis of HVSMCs were detected by CCK-8 assay and flow cytometry,respectively.Finally,the downstream targets of mir-223-5p were analyzed and identified using dual luciferase reports.Results A total of 39 DE-miRNAs were detected between TAO patients and healthy controls,among which miR-223-5p was one of the most significantly up-regulated miRNAs.Plasmaderived exosomes or miR-223-5p mimics from TAO patients can reduce cell viability of HVSMCs and promote their apoptosis,while miR-223-5p inhibitors can weaken the inhibitory activity and pro-apoptotic effect of plasma-derived exosomes from TAO patients.In addition,the expressions of vascular endothelial cell adhesion molecule-1(VCAM-1)and insulin-like growth factor-1 receptor(IGF-1R)were downregulated by plasma exosomes and miR-223-5p mimics from TAO patients,and its down-regulation was inhibited by miR-223-5p inhibitors.Dual luciferase reports suggested that VCAM-1 was the target of miR-223-5p.Conclusion Plasma derived exosomes from TAO patients may inhibit cell viability and promote apoptosis of HVSMCs through miR-223-5p/VCAM-1 pathway,which plays a role in the occurrence and development of TAO.

关 键 词：血浆外泌体 miRNA 血管平滑肌细胞(HVSMC) 生物信息学 血栓闭塞性脉管炎(TAO) 

分 类 号：R654.4[医药卫生—外科学]