人胎肝干细胞的体外分离培养与鉴定  

Isolation, culture and identification of human fetal liver stem cells In vitro

作　　者：毛海洲[1] 陈耀凯[1] 王宇明[1] 张磊[1] 刘国栋[1] 

机构地区：[1]中国人民解放军第三军医大学西南医院全军感染病研究所,重庆市400038

出　　处：《世界华人消化杂志》2005年第4期452-455,共4页World Chinese Journal of Digestology

基　　金：国家自然科学基金项目;No.30100080;No.30370391~~

摘　　要：目的:对人胎肝干细胞进行体外分离、培养与鉴定, 为其在生物人工肝及肝细胞移植方面的应用奠定基础方法:取16—29周龄死产人胚胎肝脏,参照本室建立的方法对胎肝干细胞进行分离.所获细胞悬液用50 g反复离心,沉淀物用F12培养液悬浮细胞:将细胞悬液加入Percoll液进行梯度离心,吸取50%与70%Percoll层之间的细胞层,用Hanks液洗涤两次后离心.将细胞团悬浮于DMEM/F12(2:1)培养液中,接种于塑料培养瓶或六孔培养板中;在37℃50 mL/L CO2孵箱中培养. 采用免疫组织化学法鉴定细胞表型. 结果:原代细胞呈圆形或卵圆形,体积明显小于肝细胞,胞核大而胞质少,核浆比较小,培养中细胞生长旺盛.培养的细胞胞质CK19,OV6及白蛋白染色均呈阳性;部分细胞胞质同时表达白蛋白和CK19. 结论:人胚胎肝脏中存在在肝干细胞,这些细胞表现出不同的分化状态.AIM: To isolate, culture and identify human fetal liver stem cells in vitro to lay a foundation for the application of these cells in bioartificial liver support system and hepatocyte transplantation. METHODS: The fetal liver stem cells were isolated from the liver of dead human fetus. The cell suspension was centrifuged at 50 g. The sediment was resuspended with F12 solution, followed by Percoll gradient centrifugation. The cell layer between 50% and 70% of Percoll was collected, washed, centrifuged, resuspended in DMEM (2 :1), and incubated. Immunohistochemistry was used to identify the phenotypes of the cultured cells. RESULTS: The primary cells were round or oval, much smaller than hepatocytes with larger nucleus and less plasma. These cells grew prosperously in culture medium. The plasma of the cultured cells was positive for CK19, OV6 and albumin staining. Double staining showed that some cells were positive for both albumin and CK19. CONCLUSIONS: The liver stem cells exist in the human fetal liver and display various differentiation status.

关 键 词：胎肝 肝干细胞 人胎 CK19 人胚胎 体外分离 肝脏 细胞悬液 DMEM 悬浮细胞 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]