尼古丁对人外周血内皮干细胞数量和活性的影响

Effect of nicotine on number and activities of human endothelial progenitor cells

目的 观察尼古丁影响出生后的血管新生是否与其影响外周血内皮干细胞(EPC)的数量和活性有关。方法 采用密度梯度离心法从外周血获取单个核细胞,将其接种在人纤维连接蛋白包被培养板,培养7d后,收集贴壁细胞,加入不同浓度尼古丁(分别为10 - 12 ,10 - 10 ,10 - 8,10 - 6 及10 - 4mol·L- 1)培养一定的时间(12 ,18,2 4 ,32及4 8h)。激光共聚焦显微镜鉴定FITC UEA Ⅰ和DiI acLDL双染色阳性细胞被认为是正在分化的EPC ,流式细胞仪检测其表面标志进一步鉴定EPC ,倒置荧光显微镜下计数。然后分别采用MTT比色法、改良的Boyden小室、体外血管生成试剂盒和粘附能力检测实验,观察EPC的增殖能力、迁移能力、体外血管生成能力和粘附能力。结果 尼古丁在浓度为10 - 12 ～10 - 8mol·L- 1范围内显著增加外周血EPC数量,改善外周血EPC的粘附能力、迁移能力、增殖能力和体外血管生成能力,并且EPC数量随尼古丁浓度增加而增加,10 - 8mol·L- 1浓度尼古丁EPC影响最为显著(P <0 .0 1,n =6 )。另外,10 - 8mol·L- 1尼古丁呈时间依赖地增加外周血EPC数量,改善外周血EPC功能。而尼古丁在浓度>10 - 6 mol·L- 1却减少外周血EPC数量,损害外周血EPC功能。结论 尼古丁可增加EPC数量并改善其功能。

AIM To investigate whether nicotine has influences on human endothelial progenitor cells (EPC) number and activities, which results in postnatal neovascularization. METHODS Total mononuclear cells (MNC) were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin -coated culture dishes. After 7 d cultured, attached cells were stimulated with nicotine (final concentrations: 10 -12 , 10 -10 , 10 -8 , 10 -6 and 10 -4 mol·L -1 ) or vehicle control for the respective time points(12, 18, 24, 32 and 48 h). EPC were characterized as adherent cells double positive for DiLDL-uptake and lectin binding by direct fluorescent staining under a laser scanning confocal microscope. EPC were further documented by demonstrating the expression of KDR, VEGFR-2 and AC133 with flow cytometry . EPC proliferation, migration and in vitro vasculogenesis activity were assayed with MTT assay, modified Boyden chamber assay and in vitro vasculogenesis kit, respectively. EPC adhesion assay was performed by replating those on fibronectin-coated dishes, and then adherent cells were counted. RESULTS Incubation of isolated human MNC with nicotine concentration-dependently increased EPC number, proliferative, migratory , adhesive activity and in vitro vasculogenesis at nicotine concentrations of 10 -12 -10 -8 mol·L -1 . The peak effects on EPC were observed at concentrations of nicotine 10 -8 mol·L -1 . In addition, nicotine (10 -8 mol·L -1 ) time-dependently increased EPC number and activity. However, cytotoxicity was seen at high nicotine concentrations (>10 -6 mol·L -1 ). CONCLUSION Nicotine has complex effects on EPC, including proliferation and activities at nicotine concentrations of 10 -12 -10 -8 mol·L -1 . However, higher nicotine concentrations induces cytotoxicity.