人脐静脉内皮细胞分离与冻存技术及Gore-Tex人工血管内皮化  

Endothelialization of Gore-Tex vascular graft by using cryopreserved human umbilical endothelial cells

作　　者：蒋红军[1] 贾兵[1] 陈张根[1] 王克强 

机构地区：[1]复旦大学附属儿科医院心血管中心外科,上海博士生200032 [2]中山医院心血管病研究所

出　　处：《中华胸心血管外科杂志》2005年第5期282-285,共4页Chinese Journal of Thoracic and Cardiovascular Surgery

摘　　要：目的体外分离人脐静脉内皮细胞,进行冷冻保存,并采用冻存脐静脉内皮细胞,复融后体外扩增培养,种植于Gore-Tex表面,进行人工血管内皮化。方法脐静脉内灌注消化液分离内皮细胞,所获内皮细胞体外培养,经免疫荧光染色及扫描电镜检查,鉴定所获内皮细胞及其纯度;采用梯度降温法,冻存内皮细胞,经复苏后,进行形态学检查,以台酚蓝试验、四氮唑盐(MTT)试验、流式细胞术细胞凋亡检测等方法鉴定细胞生物特性;冻存脐静脉内皮细胞体外培养扩增后,种植于Gore-Tex材料上,并行扫描电镜观察。结果血管内灌注消化液法可获取高纯度的内皮细胞;与新鲜细胞相比,复苏的内皮细胞活力保持在95%以上;复苏后的内皮细胞HE染色及生长曲线与新鲜细胞差异无统计学意义。冻存内皮细胞的凋亡率较新鲜内皮细胞高,但无统计学差异。冻存内皮细胞成功种植于Gore-Tex材料上,细胞生长良好,覆盖于大部分Gore-Tex表面。结论冻存内皮细胞作为种子细胞对人工血管内皮化具有可行性。脐静脉灌注酶消化法可获取足够数量及纯度的内皮细胞。冻存的内皮细胞复苏后无明显形态学改变,并保持较高的活力及体外增殖能力,可作为种子细胞来源。Objective The feasibility of constructing endothelialized vascular graft by using eryopreserved HUVCs was studies. Methods Forty-two human umbilical cords were used in this study. HUVECs were isolated by means of filling umbilical veins with digestive enzyme solution. HUVECs were then cultured and observed. Endothelial cells were identified by yen Willebrand factor immunofluoresconco staining and scanning eleetron microscope examination. Endothelial cells were suspended in eryopreserving solution which contains 10% DMSO and 10% fetal bovine serum in M199 and eryopreserved in liquid nitrogen. Post-thawed cells and non-frozen coils proliferation was evaluated by ~ the metabolic activity of tetrazolium compound. The endothelial cell growth characteristics were determined by daily observation using phase contrast microscope. Post-thawed endothelial ceils viability was determined by trypan blue staining test. How cytometry were applied to determine the apoptosis rate of post-thawed colls. Cryopreserved endothelial coils morphological examinations such as hematoxylin and eosins staining and scanning electron microscope examination were carfled out in this study. After cell culture and amplification, cryopreserved HUVCs were seeded on the inner surface of Gore-Tex graft to construct tissue engineered vascular graft. Results Extreme high-purified endothelial cells could be isolated by infusing digestive solutions to the lumen of human umbilical veins. Compared with non-frozen endothelial cells, Post-thawed endothelial cells showed 95% of vitality. Post-thawed HUVEC growth curve was similar to non-frozen ones＇. Post-thawed HUVEC apoptesis rate （5.85±0.56） % was higher than non-frozen ones （5.34 ± 0.49） % ; however, the differenco was not statistically different. Endothelializatien of vascular graft was carried out succossfully. Cryopreserved cells on Gore-Tex surface showed a good growth trend. Conclusion Cryopreserved HUVCs may be taken as a coil choice for tissue engineering. Enough high pure endothelial cel

关 键 词：内皮 血管 细胞培养 聚四氟乙烯 流式细胞术 人脐静脉内皮细胞 GORE-TEX 人工血管内皮化 冻存技术 细胞分离 扫描电镜观察 

分 类 号：R318.0[医药卫生—生物医学工程]