【摘要】 目的：经皮冠状动脉介入治疗(percutaneous coronary intervention,PCI)是冠状动脉疾病(coronary artery disease,CAD)的主要治疗方法之一，PCI术后支架内再狭窄(in-stent restenosis,ISR)是其一种严重的并发症，然而目前缺乏有效的防治手段。本研究拟检测Ras相关蛋白1A(Ras-related protein 1A,Rap1A)在ISR患者血浆中的表达水平及肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)诱导人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)炎症损伤模型中的表达差异，并探讨Rap1A在调控TNF-α诱导的HUVECs炎症中的作用，为ISR的防治提供一个新的潜在靶点。方法：纳入2020年12月至2022年7月于中南大学湘雅医院心血管内科接受PCI支架植入术，且术后1年复查经皮冠状动脉造影(coronary arteriography,CAG)的冠心病住院患者共60例。患者入院后根据CAG诊断27例为PCI术后ISR,33例为无支架内再狭窄(non-in-stent restenosis,non-ISR)。收集2组患者的临床资料，并通过酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)测定患者血浆Rap1A水平，比较2组患者血浆Rap1A水平。在细胞实验中，通过TNF-α(10 ng/mL,24 h)构建HUVECs炎症损伤模型，采用实时反转录聚合酶链反应(realtime reverse transcription PCR,real-time RT-PCR)和蛋白质印迹法检测Rap1A、白细胞介素-6(interleukin-6,IL-6)和血管细胞黏附分子-1(vascular cell adhesion molecule-1,VCAM-1)的mRNA及蛋白质表达水平。以TNF-α处理HUVECs诱导内皮细胞炎症，并运用小干扰RNA(small interfering RNA,siRNA)敲减Rap1A，以探究Rap1A在调控TNF-α诱导的HUVECs炎症中的作用。结果：与non-ISR患者相比，ISR患者中有吸烟史(P=0.005)及合并糖尿病者比例更高(P=0.028)，糖化血红蛋白(glycosylatedhemoglobin, HbA1c)(P=0.012)、低密度脂蛋白胆固醇(low-densitylipoprotein cholesterol,LDL-c)(P=0.014)及高敏C反应蛋白(hypersensitive C-reactive protein,hs-CRP)(P=0.027)水平升高，余指标在2组间差异均无统计学意义(均P>0.05)。而ISR组的血浆Rap1A水平显著高于non-ISR组[942.14(873.28～1 133.81)μg/mL vs886.93(812.61～930.98)μg/mL;P=0.004]。单因素logistic回归分析结果显示糖尿病、LDL-c、Rap1A是ISR的危险因素(均P<0.05)。在细胞实验中，与对照组相比，以10 ng/mL TNF-α诱导24 h后，HUVECs中炎症因子IL-6、VCAM-1的m RNA及蛋白质表达水平均升高(均P<0.05)，同时Rap1A的m RNA及蛋白质水平升高(均P<0.05)。进一步通过敲减HUVECs中Rap1A基因的表达，TNF-α诱导的IL-6、VCAM-1的mRNA及蛋白质表达水平均显著降低(均P<0.05)。结论：在冠心病PCI术后ISR患者血浆及TNF-α诱导的内皮细胞炎症损伤模型中，Rap1A蛋白质水平显著升高，提示Rap1A可能是预测ISR的潜在生物标志物。而在敲减Rap1A基因表达后，TNF-α诱导的内皮细胞炎症反应水平下降，提示Rap1A可能是治疗ISR内皮细胞炎症的潜在靶点。更多还原

【Abstract】 Objective: Percutaneous coronary intervention(PCI) is one of the most important treatments for coronary artery disease(CAD). However, in-stent restenosis(ISR) after PCI is a serious complication without effective measures for prevention and treatment. This study aims to investigate the Ras-related protein 1A(Rap1A) level in ISR patients and in the tumor necrosis factor-α(TNF-α)-induced inflammatory injury model of human umbilical vein endothelial cells(HUVECs), to explore the role of Rap1A in regulating TNF-α-induced inflammation in HUVECs and to provide a new potential target for ISR prevention and treatment.Methods: A total of 60 CAD patients, who underwent PCI between December 2020 and July 2022 from the Department of Cardiovascular Medicine of Xiangya Hospital, Central South University, and re-examined coronary angiography(CAG) 1 year after the operation, were included. After admission, 27 patients were diagnosed with ISR and 33 patients were diagnosed with non-in-stent restenosis(non-ISR) according to the CAG. Clinical data were collected, and the plasma Rap1A level was determined by enzyme linked immunosorbent assay(ELISA). In cell experiments, an inflammatory injury model was established with TNF-α treatment(10 ng/mL, 24 h) in HUVECs. The mRNA and protein expression levels of Rap1A, interlukin-6(IL-6), and vascular cell adhesion molecule-1(VCAM-1) were measured by real-time reverse transcription PCR and Western blotting. Small interfering RNA(siRNA) was used to explore the role of Rap1A in regulating TNF-α-induced inflammation in HUVECs.Results: Compared with the non-ISR patients, a higher proportion of ISR patients had a history of smoking(P=0.005) and diabetes(P=0.028), and higher levels of glycosylated hemoglobin(HbA1c)(P=0.012), low-density lipoprotein cholesterol(LDL-c)(P=0.014), and hypersensitive C-reactive protein(hs-CRP)(P=0.027). The remaining projects did not show significant differences(all P>0.05). The plasma level of Rap1A in the ISR group was significantly higher than that in the non-ISR group [942.14(873.28 to 1 133.81) μg/mL vs 886.93(812.61 to 930.98) μg/mL; P=0.004]. Diabetes, LDL-c, and Rap1A were risk factors for ISR by univariate logistic regression analysis(all P<0.05). The mRNA and protein expression levels of inflammatory factors IL-6 and VCAM-1 were increased in HUVECs after 10 ng/mL TNF-α treatment for 24 h compared with the control group(all P<0.05), while the m RNA and protein levels of Rap1A were increased(both P<0.05). After inhibition of Rap1A in HUVECs, the mRNA and protein expression levels of IL-6 and VCAM-1 were significantly decreased(all P<0.05).Conclusion: The plasma Rap1A level was significantly elevated in patients with ISR, suggesting that Rap1A may be a potential biomarker for predicting ISR. In the TNF-α-induced HUVECs inflammatory injury model, the expression level of Rap1A was increased. The level of TNF-α-induced endothelial cell inflammation was decreased after inhibition of Rap1A expression, suggesting that Rap1A may be a potential target for the treatment of endothelial cell inflammation in ISR.更多还原