人脐动脉脱细胞支架的制备及其生物相容性  

Preparation and biocompatibility of decellularized human umbilical artery scaffolds

作　　者：吴春根[1] 方宁涛[1] 潘銮凤[1] 

机构地区：[1]复旦大学上海医学院分子生物学实验室,上海市200032

出　　处：《中国组织工程研究与临床康复》2007年第26期5082-5085,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：目的:制备脱细胞人脐动脉支架并检测其与内皮细胞的相容性。方法:实验于2005-11/2006-12在复旦大学上海医学院分子生物学实验室完成。实验材料:健康新生儿的新鲜脐带(产妇知情同意并自愿捐献)。实验方法:①脱细胞脐动脉支架的制备:分离脐动脉放于离心管中,先用低渗后用等渗的十二烷基硫酸钠处理,再用核酸酶消化。脱细胞脐动脉支架作苏木精-伊红染色、Movat五色法染色及透射电镜观察。②支架与内皮细胞共培养:将脱细胞脐动脉支架切成小块,移入24孔培养板中。实验组每孔接种1.5mL的脐静脉内皮细胞悬液,对照组未种细胞,只加1.5mL的培养液,1周后进行扫描电镜观察。实验评估:①脱细胞脐动脉支架的形态。②血管内皮细胞在脱细胞脐动脉支架上黏附生长情况。结果:①脱细胞脐动脉支架的形态:光镜下对照组样品中观察到深蓝色的细胞核,实验组细胞结构被完全破坏,核破碎后被彻底清除,未见核碎片,血管壁纤维组织染成红色,呈整齐排列,组织结构未见明显的疏松;Movat五色法染色显示,脱细胞血管无红色的细胞质和黑色的细胞核,只见呈现整齐排列的波浪状黑色的弹性纤维,其间散布着蓝色的糖蛋白和网状纤维;透射电镜下观察到交错排列的胶原纤维和弹性纤维,有少量碎片存在,未见细胞样结构。②血管内皮细胞在脱细胞脐动脉支架上黏附生长情况:脐静脉内皮细胞种植于脱细胞脐动脉支架后,扫描电镜观察到可黏附生长并形成完整内膜层。结论:用十二烷基硫酸钠和核酸酶联合消化人脐动脉能够制备良好的脱细胞血管支架,并且对内皮细胞有良好的组织相容性。AIM： To prepare the decellularized human umbilical artery scaffold and detect its biocompatibility with human endothelial cells. METHODS： The experiment was carried out at Laboratory of Molecular Biology, Shanghai Medical College, Fudan University from November 2005 to December 2006. ①After the informed consent was obtained, human umbilical artery was isolated from one healthy neonate, put in a centrifuge tube, and treated with sodium dodecyl sulfate （SDS） in hypotonic and isotonic solution. After fully washed, the artery was digested with DNase and RNase to remove nucleic acids further. The decellularized artery then was subjected to HE staining, Movat pentachrome staining and observation under transmission electron microscope. ②The scaffold was cut into pieces and put into a 24-well culture plate. Then human umbilical vein endothelial cells （HUVECs） suspension was seeded with 1.5 mL per well in the experiment group, and the control group was not seeded with cells but only given 1.5 mL culture fluid. One week later, the morphous of the decellularized umbilical artery scaffold and the attachment of HUVECs on the scaffold were examined by scanning electron microscope. RESULTS： ①Under light microscope, sapphire nuclei were found in the control group, and in the experiment group, the cell structure was completely damaged without nuclei segments, and vessel fibrous tissue was found stained red, and regulady arranged without obvious loose structure; Movat pentachrome staining showed that there was no red cytoplasm or black nuclei in decellularized vessels, only regularly arranged black elastic fibers of wave-shape were observed, among which there were scattered blue glucoprotein and reticular fibers; under transmission electron microscope, interdigitating collagen fibers and elastic fibers were observed, accompanying with less segments, and no cell-like structure was found. ②HUVECs proliferated and formed a confluent layer on the decellularized artery scaffold. CONCLUSION： By SDS and nuclease d

关 键 词：血管组织工程 脱细胞血管支架 十二烷基硫酸钠 人血管内皮细胞 

分 类 号：R318.08[医药卫生—生物医学工程]