紫绀型先天性心脏病病人循环内皮祖细胞的数量及功能变化

目的 探讨紫绀型先天性心脏病病人外周血内皮祖细胞(endothelial progenitor cell,EPC)的数量及功能特点.方法 选取法洛四联症及单纯室间隔缺损病人各10例,采用Ficoll密度梯度离心法从外周血中分离单个核细胞,用添加牛脑垂体提取物和胎牛血清的M199培基进行体外诱导分化培养.用免疫组化和透射电镜行EPC表型鉴定;采用流式细胞仪计数CD133+/KDR+细胞;采用改良的Boyden小室,黏附能力测定实验和MTT比色法,观察EPC的迁移、黏附及增殖能力.结果 紫绀型先天性心脏病病人外周血内皮祖细胞的数量增多[集落记数(14.7±3.1)/100倍视野对(8.2±1.3)/100倍视野;DiIAcldl+/UEA-Ⅰ+细胞数(72.2±9.73)/200倍视野对(51.2±3.83)/200倍视野;CD133+/KDR+细胞百分比(0.66±0.20)%对(0.18±0.08)%,P＜0.01],其迁移能力[(140.6±9.24)/200倍视野对(91.84±8.58)/200倍视野]、黏附能力[(149.00±11.58)/200倍视野对(112.6±7.02),200倍视野]及增殖能力(OD值0.34±0.02对0.27±0.01)增强(P＜0.01).结论 紫绀型先天性心脏病病人循环内皮祖细胞的数量增多,其迁移、黏附及增殖能力增强.

Changes of circulating endothelial progenitor cells in patients with cyanotic congenital heart diseases

Objective To investigate alterations of endothelial progenitor cells (EPCs) from peripheral blood in patients with cyanotic congenital heart diseases.Methods 20 ml venous blood was obtained from patients with tetralogy of Fallot (TOF) and ventricular septal defect (VSD).The mononuclear cells were isolated by Ficoll density gradient centrifugation and cultured in Medium 199 containing 20 % fetal bovine serum (FBS) and bovine pituitary exrtract (BPE) for expansion and differentiation.The expression of EPC-specific antigens on cell surface,such as CD133,KDR,CD34,CD31 and vWF were analyzed by immunocytochemistry after 10 days.Cultured cells were further characterized by uptake of Dil-AcLDL,lectin staining by UEA-1,and flow cytometry for quantification of CD133+/KDR+ cells. Transmission electron microscopy was used to identify distinctive organelles of EPCs,namely immature mitochondria and weibel-Palade bodies.Modified Boyden chamber assay and MTT assay was used to measure the migration and proliferation of EPCs.The adhesion assay was performed by replating on fibronectin-coated dishes and then quantifying the number of adherent cells.Results The number of EPCs was significantly increased in patients with TOF compared with that of control subjects [Colony numbers (14.7±3.1) vs.8.2±1.3]/×100 field,DiI-AcLDL+/UEA-I+ (72.2±9.7) vs.(51.2±3.8)/×200 field,CD133+/KDR+ cells (0.66±0.20)% vs.(0.18±0.08)%,P＜0.01).The functional activity of EPCs such as proliferation [Optical Density (OD) value 0.34±0.02 vs. 0.27±0.01],migration[(140.6±9.2) vs.(91.8±8.6)/×200 field)] and adhesive capacity [(149.0±11.6)vs.(112.6±7.0)/×200 field)] was also significantly higher in patients with TOF.Conclusion EPC quantity and functional activity are significantly increased in patient with cyanotic congenital heart diseases.