紫绀型先天性心脏病患儿外周血内皮祖细胞的体外分离培养和鉴定

In vitro isolation,culture and identification of peripheral blood endothelial progenitor cells in children with cyanotic congenital heart disease

背景:一些基础研究成果对于新生血管形成和内皮祖细胞的自动募集机制已做出了合理的解释,体外培养的内皮祖细胞可分化为成熟的内皮细胞,有助于血管内皮修复。目的:拟建立从紫绀型先天性心脏病患儿外周血获取内皮祖细胞的方法。设计、时间及地点:细胞学体外观察,于2007-08/2008-04在中南大学湘雅二医院完成。材料:外周血来源于2007-10/12中南大学湘雅二医院胸外科收治的15例法洛四联症患儿,安静状态下末梢氧饱和度为(78.0±5.0)%。方法:采用Ficoll密度梯度离心法从外周血中分离单个核细胞,以含体积分数为0.2的胎牛血清、4mg/L牛脑垂体提取物、2mmol/L谷氨酰胺、100U/mL氨苄青霉素、100g/L硫酸链霉素的M199培养基进行体外诱导分化,调整细胞密度为2×109L-1,接种至预铺人纤维连接蛋白的6孔培养板,置于37℃、体积分数为0.05的CO2饱和湿度条件下,贴壁法纯化培养10d。主要观察指标:细胞形态和生长情况,Dil标记的乙酰化低密度脂蛋白/FITC标记的荆豆凝集素双荧光染色鉴定结果,vWF、CD34及CD31免疫组化鉴定结果,PE标记CD133抗体与藻蓝蛋白标记KDR抗体免疫荧光鉴定结果,透射电镜观察内皮祖细胞特征细胞器。结果:接种后0.5h细胞贴壁生长,培养第7天形成典型的细胞集落,第14天呈铺路石样外观,细胞数量级可达108。培养第10天,贴壁细胞中70%呈Dil标记的乙酰化低密度脂蛋白/FITC-UEA-Ⅰ双阳性,为正在分化的内皮祖细胞;vWF、CD34及CD31免疫组化染色均呈阳性表达;PE标记CD133抗体呈红色荧光,藻蓝蛋白标记KDR抗体呈蓝色荧光。透射电镜可见内皮祖细胞特征性幼稚线粒体及Weibel-Palade小体。结论:实验成功从紫绀型先天性心脏病患儿外周血中分离培养并获取高纯度的内皮祖细胞,且在体外稳定快速扩增。

BACKGROUND： Some basic researches have confirmed the mechanism of neovascularization and automatic recruitment of endothelial progenitor cells. In vitro cultured endothelial progenitor cells can differentiate into mature endothelial cells, which is beneficial for endothelial repair. OBJECTIVE： To establish methods of harvesting endothelial progenitor cells from peripheral blood in children patients with cyanotic congenital heart disease. DESIGN, TIME AND SETTING： The cytology in vitro observation was performed at the Second Xiangya Hospital, Central South University from August 2007 to April 2008. MATERIALS： Peripheral blood was obtained from fifteen tetralogy of Fallot patients at the Department of Cardiothoracic Surgery, Second Xiangya Hospital, Central South University from October to December 2007. Peripheral oxygen saturation was （78.0±5.0）% under a resting state. METHODS： Mononuclear ceils were isolated by Ficoll density gradient eentrifugation. The isolated ceils were suspended in Medium 199, supplemented with 20% fetal bovine serum, 4 mg/L bovine pituitary extract, 2 mmol/L glutamine, 100 U/mL ampicillin and 100 g/L streptomycin sulfate for culturing, differentiating and proliferating. At a density of2×10^9 L^-1, cells were incubated in a six-well culture plate covered by human fibronectin under saturated humidity of 0.05 volume fraction CO2 at 37℃, and cultured by adherence method for 10 days. MAIN OUTCOME MEASURES： Cell morphology, growth; Cultured cells were further stained with Dil-acetylation low density lipoprotein （AcLDL） and FITC-ulex europaeus agglutinin （UEA）-1 using double fluorescence staining. The expressions of specific antigens on cell surface, such as vWF, CD34 and CD31 were analyzed by immunocytochemistry. PE labeled CD133 antibody and phycocyanin labeled KDR antibody were measured by immunofluorescence. Transmission electron microscope was used to identify the distinct cell organelles of endothelial progenitor cells. RESULTS： Cells adhered at 0.5 hour after incubation; Typical cell colony was formed at 7 days; Cells presented paving stone-shape at 14 days in total number of 108. At day 10, 70% adhered cells were positively expressed for Dil-AcLDL and FITC-UEA-I, which were endothelial progenitor cells. A positive reaction to immunohistochemistry was detected in vWF, CD34 and CD31. PE labeled CD 133 antibody showed red fluorescence, whereas phycocyanin-labeled KDR antibody displayed blue fluorescence. The specialized immature mitochondrion and Weibel-Palade body could be found in endothelial progenitor cells under a transmission electron microscope. CONCLUSION： Endothelial progenitor cells with a high purity can be obtained from mononuclear cells in the peripheral blood of cyanotic congenital heart disease patients, and can proliferate rapidly and stably in vitro.