血浆微小RNA作为下肢动脉硬化闭塞症介入治疗后再狭窄生物标记物的研究

Lower extremity arteriosclerosis obliterans is a severe vascular disease with high morbidity.A timely and accurate diagnosis for in-stent restenosis after endovascular treatment of lower extremity arteriosclerosis obliterans is the key to reduce serious complications. MicroRNAs (miRNAs) are small regulatory RNAs that have shown promise as tissue-based markers for cardiovascular disease classification and prognostication. Our previous studies show that miRNAs（miR-320a，miR-572 etc.） have specific expression in the intima of in-stent restenosis after endovascular treatment of lower extremity arteriosclerosis obliterans. We hypothesized that miRNAs could be an ideal class of blood-based biomarkers for detection of in-stent restenosis after endovascular treatment of lower extremity arteriosclerosis obliterans. In this study, microarray was performed on plasma of controls and in-stent restenosis after endovascular treatment of lower extremity arteriosclerosis obliterans patients. Completing the statistical analysis for significance and prediction, we selected candidate miRNAs for a second large study by RT-qPCR in controls and whom were diagnosed with in-stent restenosis after endovascular treatment of lower extremity arteriosclerosis obliterans. Additionally, we combined specific miRNA with different stages and subtypes of in-stent restenosis after endovascular treatment of lower extremity arteriosclerosis obliterans to complete the integrative analysis. Finally, we explore the histology of specific miRNA through animal experiments, and assess the stability of miRNA. Given this, we speculate that miRNAs could have value in the setting of detecting in-stent restenosis after endovascular treatment of lower extremity arteriosclerosis obliterans for which clinically validated blood biomarkers are lacking.These biomarkers provide important clues in the early diagnosis of in-stent restenosis after endovascular treatment of lower extremity arteriosclerosis obliterans.

下肢动脉硬化闭塞症严重影响患者的生活质量甚至危及生命，对于介入术后再狭窄的早期预警并采取合理的治疗可大幅降低严重并发症。循环血中微小RNA是一类新近研究发现具有分子诊断标志物优点的内源性小分子核苷酸序列。本项目组前期研究表明再狭窄患者术前血浆中存在微小RNA的特异改变，通过高通量技术初步筛选出一组再狭窄患者循环血中差异微小RNA（miR-320a，miR-572等）；那么循环血中微小RNA是否可以作为下肢动脉硬化闭塞症术后再狭窄的生物标记物呢？本研究拟：①针对芯片筛选结果进行芯片显著性分析和预测分析以获得与疾病密切相关的特定微小RNA；②针对特定微小RNA进行大样本的定量验证,结合临床分型、病程转归及预后进行综合分析，建立疾病早期预警模型并对其进行独立样本验证和校正；③通过动物实验探索特定微小RNA的组织学基础，并对其进行稳定性评价。以期为再狭窄的早期预警寻找临床可用的实验室检查指标。

摘要.背景：支架内再狭窄（ISR）仍然是下肢动脉硬化闭塞症（LEAOD）患者血管内支架成形术后失败的重要原因之一。早期诊断应选用敏感可靠的生物标志物来预测支架内再狭窄的发生。本文旨在探讨microRNA对下肢动脉硬化闭塞症患者经血管内支架成形术后支架内再狭窄的预测价值。.资料与方法：选取2014年3月至2016年7月在我科接受腔内治疗的208例LEAOD患者(其中男性170例，女性38例)。根据术后常规血管造影将患者分为支架术后再狭窄组(ISR)和无再狭窄组(non-ISR)。检测208名受试者中循环血中的microRNA表达情况，ISR患者78例，non-ISR患者68例，健康志愿者62例。我们通过基因芯片筛选出6种microRNA作为候选基因并通过qRT-PCR进一步验证相关的候选基因。通过ROC曲线评价循环血中的microRNA对LEAOD患者ISR的预测价值。.结果：结果显示，在ISR组中，循环血中microRNA-320a和microRNA-572 (n = 78)表达水平明显高于non-ISR组和健康志愿者组。通过ROC曲线分析，microRNA-320a的敏感性为82.1%，特异性为63.8%；microRNA-572的敏感性为69.2%，特异性为68.9%，它们的ACU分别为0.766和0.690。此外，相较于68例non-ISR患者和62例健康志愿者，78例ISR患者的microRNA-3937和microRNA-642a-3p循环表达水平明显较高，而microRNA-4669和microRNA-3138循环表达水平较低，但差异无统计学意义。.结论：研究发现ISR患者循环血中microRNA表达增高，提示microRNA可作为一种有效预测LEAOD患者ISR的预测因子。这些数据表明，循环血中microRNA-320a和microRNA-572在LEAOD患者的ISR诊断中具有很好的预测价值。