人脐静脉内皮细胞制备饲养层对胚胎干细胞生长的支持作用  

Human Umbilical Venous Endothelial Cells as Feeder Layer to Support the Growth of Embryonic Stem Cells

作　　者：何志旭[1] 王志华[1] 米蔷[1] 汪浩文[1] 

机构地区：[1]贵阳医学院附属医院儿科/贵阳医学院干细胞研究中心,贵阳550004

出　　处：《实用儿科临床杂志》2008年第19期1526-1529,共4页Journal of Applied Clinical Pediatrics

基　　金：教育部新世纪优秀人才支持计划项目资助(050820);贵州省高校发展专项资金项目资助(黔教科2004114);贵州省省长资金项目资助(200614)

摘　　要：目的探讨人脐静脉内皮细胞是否可作为饲养层支持胚胎干细胞(ESC)的生长,维持ESC的未分化状态。方法取健康产妇产后的脐带,胶原酶灌注消化脐静脉血管,分离内皮细胞原代培养,并连续传代扩增细胞,Ⅷ因子相关抗原鉴定。采用生长良好的3代以上内皮细胞,经丝裂霉素C(10mg/L)灭活后制备饲养层,将E14.1 ESC接种到该饲养层上进行传代培养,观察ESC的形态学特征,测定不同传代时间的ESC碱性磷酸酶(AKP)活性、干细胞标志物Oct-4的表达,严重联合免疫缺陷(SCID)小鼠体内畸胎瘤形成实验鉴定其全能分化特性。结果人脐静脉血管来源的内皮细胞能够在体外培养过程中生长、增殖旺盛,细胞连续传代10代以上能完整的保持正常的细胞形态学特征和生物学特性。经丝裂霉素C处理后细胞停止增殖,在24h内能较好的保持细胞活力和形态学特征,具备饲养层细胞的基本条件。E14.1 ESC在人脐静脉内皮细胞上传3～8代能较好的保持未分化状态,细胞呈克隆性生长,高度表达AKP及干细胞因子Oct-4;体内全能分化实验显示:在内皮细胞上生长6代和10代以上的E14.1 ESC接种到SCID小鼠6周后均能形成畸胎瘤,含有3个胚层的组织细胞。结论人脐静脉血管内皮细胞可作为一种来源丰富,取材方便的人源化饲养层细胞,能有效支持ESC的生长,克服了目前人ESC使用动物细胞饲养层带来的异种蛋白污染和致病微生物的危险,解决了ESC临床应用的生物安全性问题。Objective To explore whether human umbilical vennus endothelial cells could be used as feeder later to suppnrt the growth of embryonic stern cells （ESC） and keep ESC undifferentiated. Methods The venous vessels of umbilical cord obtained from healthy puerperal were perfused with collagenase. The isolated endothelial cells went through primary culture and passages for expansion. Factor Ⅷ antigens determintion was implemented. Endothelial cells with good growth and 3 or above passages were treated with mitomyein - C（ 10 mg/L） and prepared as feeder layer,on which E14.1 ESC was transplanted for subculturing to observe the morphological characterization and determine ESC alkaline psphatase （AKP） activity anti the expression of stem cell marker Oct -4. Severe combined immune deficiency （SCID） mouse in vivo terotoma formatinn experiment was performed to identify its pluripntent properties. Results Human umbilical vein - derived endothelial cells grew well in culturing in vitro and regenerate in large numbers. The endothelial cells maintained normal cellular morphologieal and biological eharaeterization after 10 passages. The cells stopped proliferating after being treated with, mitomyein - C, but its activity and morphological properties were well -maintained with 24 hours,which was a fundamental property of serving as feeder layer. E14.1 ESC remained undifferentiated in haman umbilical venous endothelial cells after 3 -8 passages,the cells grew in colony and showed high expression of AKP and stem cell Oct -4. In vivo pluripntency experiment showed that 6 weeks after being transplanted to SCID mice E14. 1 ESC of 6 and 10 passages in endothelial cells both could form teratoma containing 3 layers of tissue cells. Conclusions Human umbilical venous endothelial cell serve as a convenient feeder laver cell with rich sources. It can effectively support ESC growth and heterogenous and prevent the heterogeneous protein pnllutinn and pathogenic microorganisms caused by animal cell feeder layers,thus solve the

关 键 词：胚胎干细胞 脐静脉 内皮细胞 饲养层 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]