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目的观察急性心肌梗死再灌注对血管内皮祖细胞(EPCs)有关功能基因的影响。方法对急性前壁心肌梗死的患者心肌梗死发生6 h内和急诊经皮冠状动脉介入治疗(PCI)后7天分别采取外周血。Ficoll密度梯度离心法分离出单个核细胞(MNCs),在包被有明胶的培养瓶内以M- 199培养液培养并在刺激因子作用下,14天后收集贴壁细胞,经鉴定为EPCs。抽提RNA,进行24个内皮相关基因在心肌梗死即刻和再灌注治疗7天后表达差异的芯片检测,并用RT-PCR的方法进行验证。24个内皮相关基因分别为调节血管收缩舒张功能、血管新生和内皮细胞活性的相关基因: ACE、AGTR-1、AGTR-2、eNOS、COX-2、ET-1、ETA、ETB、SOD-1、CDH5、VEGF、VEGFR1、VEGFR2、ICAM1、ICAM2、ICAM3、PECAM-1、E-Selectin、L-Selection、VCAM1、tPA,uPA,PAI,vWF。同时,收集25例进行急诊PCI的急性心肌梗死患者即刻和7天的外周抗凝血,用流式细胞检测法比较其外周血中一些带有内皮功能标志物的细胞数量变化(如:VEGFR2、PECAM-1、VE-cadherin等)。结果基因芯片检测结果显示再灌注7天后(Cy3)较心肌梗死6 h内(Cy5)有8个基因的表达降低(cy3／cy5≤0．05),分别为AGTB-1、AGTR-2、COX-2、eNOS、ET-1、ETA、VEGF。其余17个基因无差异(Cy3／Cy5在0．05-2．00之间)。对差异表达基因AGTR-1和ETA及无差异表达基因SOD-1进行BT-PCR验证也显示了相同结果。流式细胞检测显示再灌注治疗后7天外周血中VEGFR2阳性细胞显著下降(P< 0．05),PECAM-1和VE-cadherin阳性细胞无明显变化(P>0．05)。结论根据以上实验结果推测再灌注治疗后早期能使EPCs在血管舒缩方面的某些功能异常得到恢复。另外,由于缺血的改善EPCs血管新生作用减弱,同时局部的炎症反应也会减轻,有可能进一步稳定冠状动脉的病变。
Objective To observe the effects of reperfusion on the functional genes of endothelial progenitor cells (EPCs) in acute myocardial infarction (AMI). Methods Peripheral blood was collected within 6 hours after acute myocardial infarction (AMI) and within 7 days after emergency percutaneous coronary intervention (PCI). Mononuclear cells (MNCs) were isolated by Ficoll density gradient centrifugation and cultured in M-199 culture medium in gelatin-coated flasks. After 14 days, adherent cells were collected and identified as EPCs. RNA was extracted and 24 endothelium-related genes were detected for chip expression difference immediately after myocardial infarction and 7 days after reperfusion, and verified by RT-PCR. Twenty-four endothelium-related genes were ACE, AGTR-1, AGTR-2, eNOS, COX-2, ET-1, ETA, ETB and SOD- 1, respectively, which regulate vasoconstriction and diastolic function, angiogenesis and endothelial cell activity , CDH5, VEGF, VEGFR1, VEGFR2, ICAM1, ICAM2, ICAM3, PECAM-1, E-Selectin, L-Selection, VCAM1, tPA, uPA, PAI, vWF. At the same time, 25 patients with acute myocardial infarction (PCI) were enrolled in this study. Immediate and 7-day peripheral anticoagulant blood samples were collected. The number of cells with endothelial function markers in peripheral blood was compared by flow cytometry (such as VEGFR2, PECAM-1, VE-cadherin, etc.). Results The results of microarray assay showed that the expression of 8 genes in Cy5 was significantly lower than that in Cy5 (Cy3 / cy5≤0.05) at 7 days after reperfusion (Cy3), which were AGTB-1, AGTR-2, -2, eNOS, ET-1, ETA, VEGF. The remaining 17 genes were not different (Cy3 / Cy5 between 0.05-2.00). BT-PCR verification of the differentially expressed genes AGTR-1 and ETA and the undifferentially expressed gene SOD-1 also showed the same result. Flow cytometry showed that the number of VEGFR2 positive cells in peripheral blood decreased significantly (P <0.05) 7 days after reperfusion, but there was no significant change in PECAM-1 and VE-cadherin positive cells (P> 0.05). Conclusions According to the above experimental results, it is speculated that some functional abnormalities of EPCs in vasomotor may be recovered early after reperfusion. In addition, due to ischemic improved EPCs angiogenesis weakened, while the local inflammatory response will be reduced, it is possible to further stabilize the coronary lesions.