循环miR-500对内皮祖细胞募集和颈动脉粥样斑块稳定性的调节作用

Regulatory role of circulating mi R-500 in recruitment of endothelial progenitor cells and stability of carotid atherosclerotic plaque

目的探讨mi R-500对小鼠颈动脉粥样硬化斑块中内皮祖细胞(endothelial progenitor cells,EPCs)的募集和斑块稳定性的调节作用。方法采用颈动脉硅胶圈植入法建立颈动脉粥样硬化斑块小鼠模型,实时定量聚合酶链式反应(PCR)检测正常小鼠和模型小鼠血浆中mi R-500的表达水平。32只模型小鼠随机均分为4组,每周分别尾静脉注射mi R-500模拟物、对照模拟物、mi R-500抑制物和对照抑制物,6周后收集颈动脉标本,流式细胞术检测斑块内EPCs的数量,免疫组化检测患处内皮完整性和单核/巨噬细胞浸润程度,油红O染色检测不稳定斑块面积。分离培养骨髓EPCs,分别转染mi R-500模拟物、对照模拟物、mi R-500抑制物和对照抑制物,72 h后酶联免疫吸附试验法检测炎症标志因子基质金属蛋白酶(matrix metalloprotein,MMP)-9和白细胞介素(interleukin,IL)-6的分泌,Transwell法检测细胞迁移能力。结果模型小鼠血浆中mi R-500的表达水平显著上调(P<0.01);循环mi R-500水平的减少显著促进斑块内EPCs的聚集和内皮的完整性,减少不稳定斑块的形成和斑块内单核/巨噬细胞浸润(P<0.01),抑制EPCs分泌MMP-9和IL-6,并增强其迁移能力(P<0.01);而mi R-500水平增加则作用相反。结论循环mi R-500可抑制颈动脉粥样斑块中EPCs的募集和斑块的稳定性。

Objectives To explore the role of miR- 500 in regulation of recruitment of endothelial progenitor cells（EPCs）and stability of carotid atherosclerotic plaque in mice. Methods The carotid atherosclerotic plaque mousemodel was established by carotid artery silicone rubber ring implantation. The expression level of miR-500 in plasma ofnormal mice and model mice was detected by real-time quantitative polymerase chain reaction（PCR）. The 32 modelmice were randomly divided into 4 groups and respectively intervened with miR-500 mimic,control mimic,miR-500inhibitor and control inhibitor by intravenous injection every week. After 6 weeks,carotid artery specimens were collected,flow cytometry was used to measure the number of EPCs in the plaque,immunohistochemistry was used to detect endo.thelial integrity and the infiltration of monocytes/macrophages,oil red O staining was used to detect the size of unstableplaque. Then,bone marrow derived EPCs were isolated,and miR-500 mimic,control mimic,miR-500 inhibitor andcontrol inhibitor were respectively transfected into EPCs. After incubation for 72 h,the secretion of inflammatory markers（matrix metalloprotein,MMP）-9 and interleukin（IL）-6 was detected with enzyme linked immunosorbent assay,andthe capacity of cell migration was detected with Transwell cell migration assay. Results MiR-500 was significantlyupregulated in the plasma of model mice（P〈0.01）. Inhibition of circulating mir-500 promoted EPC recruiment andendothelial integrity,reduced the formation of unstable plaque and accumulation of monocytes/macrophages（P〈0.01）,inhibited the secretion of MMP-9 and IL-6,and enhanced the capacity of cell migration（P〈0.01）. In contrast,upreg.ulation of mir-500 had an opposite effect. Conclusions Circulating miR-500 suppresses the recruitment of EPCs andthe stability of carotid atherosclerotic plaque.