急性心肌梗死再灌注后近期血管内皮祖细胞相关功能基因的变化

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6h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、VCAMl、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6h内（Cy5）有8个基因的表达降低（Cy3／Cy5≤0．05）,分别为AGTR-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RT-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。

Endothelial progenitor cells related gene expression changes before and early after revascularization in patients with acute myocardial infarction

Objective The purpose of this study was to observe the endothelial progenitor cells (EPCs) related gene expression changes before and early after revascularization in patients with acute myocardial infarction. Methods Peripheral blood samples were taken from patients with acute anterior myocardial infarction 6 hours and 7 days after PCI and stenting. Mononuclear cells ( MNCs) were isolated by Ficoll-density centrifugation and cultured in M-199 medium. After 14 days culture, attaching cells incorporated DiI-acetylated low-density lipoprotein (EPCs) were collected and RNA was isolated by Trizol for microarray analysis on 24 genes associated with permissibility/vessel tone (angiotensin system: ACE, AGTR-1, AGTR-2; NO system: eNOS; prostacyclin system: COX-2; endothelin system: ET-1, ETA, ETB; superoxide anions system: SOD-1) , angiogenesis (adhesion molecule: CDH5; growth factors and receptors: VEGFR1, VEGFR2, VEGF) and endothelial cell activition ( adhesion molecules expression: ICAM1, ICAM2, ICAM3, PECAM-1, E-Selectin, L-Selection, VCAM1; change phenotype from antithrombotic to prothrombotic: tPA, uPA, PAI, vWF). VEGFR2, PECAM-1 and VE-cadherin positive cells were identified by flow cytometry. Result Eight gene expressions (AGTR-1, AGTR-2, COX-2, eNOS, ET-1,ETA, VEGF) were significantly downregulated 7 days post PCI compared to pre-PCI (P<0.05). Flow cytometry results showed that VEGFR2 positive cells were also significantly reduced post PCI than that of before PCI (P<0.05) . Conclusion PCI down-regulated endothelial progenitor cells related gene expressions in patients with acute myocardial infarction.