冠状动脉支架置入后再狭窄患者外周血内皮祖细胞数量及活性的变化

背景：近年来发现的内皮祖细胞能够促进受损内皮愈合,因而假设支架置入后再狭窄可能与内皮祖细胞的数量和/或活性有关。目的：对比观察冠状动脉支架置入后再狭窄患者与未发生再狭窄患者循环内皮祖细胞数量及活性,验证上述假设。设计、时间及地点：对比观察实验,于2005-03/2007-05在北京世纪坛医院心内科、北京大学第一医院心内科及北京大学医学部生理学与病理生理学系完成。对象：根据复查冠脉造影的结果将来源于北京大学第一医院、既往行冠脉支架置入的患者分为两组：发生支架置入后再狭窄组（n=15）及未发生支架置入后再狭窄的对照组（n=17）。方法：采用密度梯度离心法获取外周血总的单个核细胞,接种到包被有人纤维连接蛋白的24孔培养板,7d后通过激光共聚焦显微镜测定贴壁细胞摄取DiI-acLDL及结合FITC-UEA-Ⅰ的能力,双染色阳性的细胞为正在分化的内皮祖细胞。主要观察指标：外周血单个核细胞培养7d后,通过倒置显微镜计算内皮祖细胞的数量、通过MTT比色法测定内皮祖细胞增殖倍数、通过划痕试验定性观察内皮祖细胞的迁移数量以及通过黏附试验测定内皮祖细胞的黏附率。结果：再狭窄组内皮祖细胞数量明显低于对照组（P=0.001）,再狭窄组的内皮祖细胞增殖倍数明显低于对照组（P〈0.05）,再狭窄组的内皮祖细胞迁移数量明显低于对照组,但两组之间的内皮祖细胞黏附率差异无显著性意义（P〉0.05）。结论：冠状动脉支架置入后再狭窄患者的内皮祖细胞数量及增殖能力、迁移能力明显下降,说明支架置入后再狭窄可能与内皮祖细胞的数量和/或活性有关。

Number and activity of circulating endothelial progenitor cells in patients with coronary in-stent restenosis

BACKGROUND: It has been recently found that endothelial progenitor cells (EPCs) can promote injured endothelial healing. There is a supposition that in-stent restenosis possibly correlates with the number and/or activity of EPCs.OBJECTIVE: To comparatively observe the number and activity of circulating EPCs in patients with and without coronary in-stent restenosis, and to verify the above-mentioned supposition.DESIGN, TIME AND SETTING: This study, a comparative observation, was performed at the Department of Internal Medicine, Beijing Shijitan Hospital, Department of Internal Medicine, First Hospital, Peking University, and Department of Physiology and Pathophysiology, Peking University Health Science Center between March 2005 and May 2007.PARTICIPANTS: According to the coronary angiography, 15 patients were recruited into the restenosis group and 17patients with patent stents were selected into the control group.METHODS: Total peripheral mononuclear cells were isolated from blood of patients with restenosis or control subjects by Ficoll density-gradient centrifugation. These cells were plated on dishes coated with human fibronection. After 7 days in culture, the nature of adherent cells was confirmed by direct fluorescent staining with the use of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanide percholate-labelled acetylated low-dencity lipoprotein and fluorescein isothiocyanate-labeled ulex europaeus agglutinin-Ⅰ under a laser scanning confocal microscope. Cells demonstrating double-positive fluorescence were identified as differentiating EPCs.MAIN OUTCOME MEASURES: After 7 days of culture, EPCs were counted under an inverted microscope. Proliferation of EPCs was determined using the MTT colorimetric assay. Migration of EPCs was assayed using the scratch assay qualitatively. EPCs adhesion was performed by replating cells on fibronectin-coated dishes and then counting the adherent cells.RESULTS: The number of EPCs was significantly reduced in patients with in-stent restenosis compared with that in the control group (P = 0.001). The proliferative activities were impaired in the in-stent restenosis group than in the control group(P < 0.05). In addition, the migrative activities were also impaired in the in-stent restenosis group, but no significant difference in adherent activities existed between the two groups (P > 0.05).CONCLUSION: The number and functional activities of proliferation and migration of EPCs were decreased in patients with in-stent restenosis, which may be related to the number and/or activities of EPCs.