人胚胎干细胞无血清无饲养层培养体系的建立  

Establishment of feeder layer- and serum-free culture system of human embryonic stem cells

作　　者：胡智兴[1] 吴兰鸥[1] 郑春兰[1] 周轶平[2] 罗敏[2] 梁道明[3] 

机构地区：[1]昆明医学院药理学教研室,云南省昆明市650031 [2]昆明医学院云南省天然药物药理重点实验室,云南省昆明市650031 [3]昆明医学院第二附属医院,云南省昆明市650031

出　　处：《中国组织工程研究与临床康复》2009年第45期8889-8894,共6页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：云南省科技厅-昆明医学院应用基础研究联合专项基金(2007C0012R;2007C0044R)~~

摘　　要：背景:研究表明FGF2,TGFβ/activin/nodal和IGF信号通路是人胚胎干细胞保持其多能性所必需的,然而直接添加外源性碱性成纤维细胞生长因子、转化生长因子β和胰岛素是否能够维持人胚胎干细胞的自我更新目前尚无报道。目的:拟建立人胚胎干细胞无饲养层无血清条件下的培养体系。设计、时间及地点:细胞学体外观察,于2007-09/2009-02在中国科学院昆明动物研究所完成。材料:12.5～13.5d龄清洁级ICR孕鼠2只,由昆明医学院动物中心提供。人胚胎干细胞株BG02购自美国Bresagen公司。方法:①消化离心BG02细胞,重悬于无饲养层过渡培养基后接种,常规培养5～7d,挑去分化的人胚胎干细胞,加入分散酶消化,切割成小团块,离心重悬后按1∶3比例重新接种预铺层粘连蛋白的4孔板,此时培养基换成无血清无饲养层培养基,由80%DMEM/F12、20%KSR、2mmol/Lglutamine、1%非必需氨基酸、0.1mmol/Lβ-巯基乙醇、ITS(×1)、106U/L青霉素、100mg/L链霉素、4μg/L碱性成纤维细胞生长因子、0.12μg/L转化生长因子β1组成。②无菌条件下取出ICR胎鼠,组织块胰酶消化法分离培养小鼠胚胎成纤维细胞,接种在0.1%明胶包被的6孔板中即为饲养层。将生长在小鼠胚胎成纤维细胞饲养层上的人胚胎干细胞株BG02预铺至有层粘连蛋白的培养板上,加入含碱性成纤维细胞生长因子、转化生长因子β1及ITS的无血清培养基连续培养。主要观察指标:观察BG02细胞在无血清无饲养层培养体系中的形态,采用细胞免疫组化法检测人胚胎干细胞特异性分子标志的表达,检测BG02细胞体外分化能力及其核型,比较BG02细胞在无血清无饲养层和小鼠胚胎成纤维细胞饲养层培养体系中的生长情况、细胞集落分化率。结果:在无血清无饲养层培养体系中,BG02细胞连续传代20代,细胞呈典型的人胚胎干细胞形态特征;BG02细胞表达SSEA-4,SSEA-3,TRA-1-60,TRA-1-81,Oct-4,�BACKGROUND： FGF2, TGFβ/activin/nodal and IGF signal pathways are necessary for keeping functions of human embryonic stem cells, but there was no report concerning whether addition of exogenous basic fibroblast growth factor, transforming growth factor β and insulin can maintain self-renewal of human embryonic stem cells. OBJECTIVE： To establish a feeder layer- and serum-free culture system of human embryonic stem cells. DESIGN, TIME AND SETTING： The cytological in vitro study was performed at the Kunming Animal Institute of Chinese Academy of Sciences from September 2007 to February 2009. MATERIALS： Pregnancy 12.5 or 13.5-day ICR strain mice （clean grade） were provided by the Animal Center of Kunming Medical College. Human embryonic stem cell line BG02 was purchased from Bresagen, USA. METHODS： BG02 cells were digested, centrifuged, resuspended in feeder layer-free medium, and then incubated for 5 7 days. Differentiated human embryonic stem cells were removed. Dispase was added for digestion. Samples were cut into blocks, centrifuged, resuspended, and then incubated at 1：3 in a 4-well plate coated with laminin in feeder layer-free and serum-free medium, supplemented with 80% DMEM/F12, 20% KSR, 2 mmol/L glutamine, 1% non-essential amino acid, 0.1 mmol/L β-mercaptoethanol, insulin-transferdn-selenium （ITS） （×1）, 10^6 U/L penicillin, 100 mg/L streptomycin, 4 pg/L basic fibroblast growth factor （bFGF） and 0.12 μg/L transforming growth factor-β1 （TGF-β1）. ICR fetus mice were sterilely obtained to culture mouse embryonic fibroblasts by tissue pancreatin digestion method. These cells were incubated in a 6-well plate coated with 0.1% gelatin. Human embryonic stem cell line BG02 cultured on mouse embryonic fibroblasts feeder layers was transferred to laminin-coated plates in serum-free medium containing bFGF, TGFβ1 and ITS. MAIN OUTCOME MEASURES： The morphology of BG02 cells in feeder layer- and serum-free condition was observed. The specific molecular markers of human e

关 键 词：人胚胎干细胞 无血清 无饲养层 培养 

分 类 号：R394.2[医药卫生—医学遗传学]