脐静脉内皮细胞低密度种植构建组织工程血管  

Low density human umbilical vein endothelial cell seeding to construct tissue-engineered vessel

作　　者：孙洪亮[1] 孔登[1] 肖琳[1] 蔡文娣[1] 黄焕生[1] 耿秀芳[1] 

机构地区：[1]潍坊医学院生物化学与分子生物学实验室,山东省潍坊市261042

出　　处：《中国组织工程研究与临床康复》2008年第33期6430-6433,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：背景:种子细胞来源不足是组织工程研究领域的难题之一,如何充分有效的利用有限的种子细胞,是一个值得深入研究的课题。目的:拟验证脐静脉内皮细胞低密度种植于脱细胞猪动脉支架实现血管内皮化的可行性。设计、时间及地点:观察性实验,于2006-10/2007-04在潍坊医学院生物化学实验室完成。材料:取新鲜猪颈动脉制备脱血管支架;新鲜脐带由潍坊人民医院产科提供,用于内皮细胞的分离和培养。方法:十二烷基硫酸钠和脱氧胆酸钠联合处理获取脱细胞猪动脉支架。0.1%胶原酶灌注获取脐静脉内皮细胞,光镜和免疫组织化学鉴定。分别将细胞以2×105cm-2低密度和2×106cm-2高密度种植于支架,静态培养11d。主要观察指标:DAPI染色法连续观察内皮细胞在支架上的生长情况。第11天对支架进行苏木精-伊红染色,扫描电镜观察,ELISA检测2×105cm-2低密度和2×106cm-2高密度培养液纤维连接蛋白的表达。结果:脱细胞支架细胞成分完全去除,胞外基质保存完好;细胞呈梭形或多角形,核仁清晰,呈典型的铺路石样;免疫荧光检测可见细胞胞浆内有颗粒状红色阳性信号。采用DAPI法连续动态观察内皮细胞在支架上生长良好,贴壁较好;苏木精-伊红染色显示支架表面形成连续融合单层细胞;扫描电镜可见内皮细胞单层形成,细胞呈多角形,椭圆形。低密度种植的培养液中纤维连接蛋白的表达量较高密度种植的高,分别为(121±7.93)μg/106个细胞和(78.20±3.21)μg/106个细胞,两者相比,差异有显著性意义(P<0.05)。结论:脐静脉内皮细胞低密度种植于脱细胞猪动脉支架不仅可以实现血管内皮化,而且利于增强内皮细胞和细胞外基质黏附力,提高贴壁细胞的抗应切力。BACKGROUND： Inadequate seed cell source is one issue in tissue engineering field. Therefore, how to effectively use seed cell becomes an important subject. OBJECTIVE： To explore endothelialization of low density human umbilical vein endothelial cell （HUVEC） seeded on decellularized porcine carotid arteries scaffold. DESIGN, TIME AND SETTING： The observation was performed at the Laboratory of Biochemistry, Weifang Medical College from October 2006 to April 2007. MATERIALS： Fresh porcine carotid artery was used to make decellularized scaffold; fresh umbilical core was provided by Department of Obstetrics, Weifang people＇s Hospital for endothelial cell isolation and culture. METHODS： Decellularized scaffold was prepared by treating fresh porcine artery and heart valves with sodium deoxycholate and sodium dodecyl sulfate. The HUVECs were isolated from fresh umbilical vein with 0.1% collagenase A and were identified by cell morphology and immunostaining. The HUVECs were seeded on the scaffold at 2×10^5 cells/cm^2 and 2×10^6 cells/cm^2 and cultured for 11 days. MAIN OUTCOME MEASURES： Endothelial cell growth condition on the scaffold was observed using DAPI staining, and were statically observed by HE staining and scanning electron microscope on the 1 lth day. The expression of fibronectin in the culture medium was detected by ELISA. RESULTS： The cellular components of heart valves were completely removed and the structure of extracellular matrix was well reserved. The HUVECs were in spindle or polygon, and retained cobblestone appearance. Immunofluorescence staining showed that there were red positive plasmids in cytoplasm. DAPI staining showed that the endothelial cells on the scaffolds grew well. HE immunostaining showed a continuing endothelial cell layer on the surface. Scanning electron observation displayed that the scaffold had been reendothelializated, and cells were in polygon or oval shape. In the culture medium, the expression of fibronectin with low density seeding was significantly hi

关 键 词：纤维连接蛋白 低密度种植 脱细胞血管支架 组织构建 

分 类 号：R318[医药卫生—生物医学工程]