三种不同饲养层上人胚胎干细胞生长状态的比较(英文)  

Growth of human embryonic stem cells in three differen feeder layers

作　　者：李斌[1] 彭秋平[2] 卢伟英[1] 徐雯[1] 金应霞[2] 黄元华[1] 

机构地区：[1]海南医学院,海南省海口市571101 [2]海南医学院附属医院生殖医学中心,海南省海口市570102

出　　处：《中国组织工程研究与临床康复》2008年第8期1586-1590,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：背景：人胚胎干细胞传代培养的关键是抑制其自发分化、保证细胞的全能性。以小鼠胚胎成纤维细胞或人包皮成纤维细胞作为饲养层尽管能够维持胚胎干细胞的未分化状态，但存在细胞克隆不饱满、平铺情况明显等问题。目的：制备小鼠胚胎成纤维细胞与人包皮成纤维细胞的混合饲养层，观察人胚胎干细胞在其上面的生长状态。设计：多样本观察比较。单位：海南医学院附属医院生殖医学中心。材料：实验于2006—04／2007—07在海南医学院附属医院生殖医学中心完成。包皮来自于行包皮环切的儿童，由海南医学院附属医院泌尿外科提供，儿童家属对治疗及实验均签署知情同意书，实验经医院医学伦理委员会批准。人胚胎干细胞系HN—1由本实验室从人类囊胚中分离培养并鉴定。清洁级孕12．6-14．5d的胎鼠11只，实验过程中对动物的处置符合动物伦理学标准。方法：胎鼠麻醉后去除头、四肢和内脏，按常规胰蛋白酶反复消化法获得细胞悬液进行接种培养，待生长汇合后冻存部分原代细胞，用丝裂霉素c处理2．0—3．0h后，按1 × 10^8 L^-1密度接种于明胶包被的中心皿内，UPd,鼠胚胎成纤维细胞饲养层。人包皮成纤维细胞的分离培养与饲养层制备同上。上述两种成纤维细胞分别计数后，按1：0，3：1，1：1，1：3，0：1比例混合，然后以1 × 10^8 L^-1密度接种于明胶包被的中心皿内，即混合饲养层。观察体外传代培养的人胚胎干细胞在3种不同饲养层上的生长状态，并对生长在混合饲养层上的人胚胎干细胞进行碱性磷酸酶检测、OCT-4表达免疫组化检测、OCT-4及端粒酶mRNA表达RT-PCR检测。撤除饲养层，观察人胚胎干细胞体外分化情况。主要观察指标：①不同饲养层上人胚胎干细胞的生长状态比较。②人胚胎干细胞在不同比例混合饲养层上的生长状态比较�BACKGROUND： Key point for subculture of human embryonic stem cells （hESCs） is to inhibit spontaneous differentiation and make sure totipotency of cells. Although mouse embryonic fibroblasts （MEF） or human foreskin fibroblasts （HFF） used as the feeder layer can maintain undifferentiated state of embryonic stem cells, cell clone is still imperfect and parallel arranged. OBJECTIVE： To establish mixed feeder layer of mouse embryonic fibroblasts plus human foreskin fibroblasts and to observe the hESCs growth. DESIGN： Multi-sample observation and comparison. SETTING： Medical Center of Reproduction, the Affiliated Hospital of Halnan Medical College. MATERIALS： This study was performed at the Medical Center of Reproduction, the Affiliated Hospital of Halnan Medical College from April 2006 to July 2007. Foreskin was derived from the children who underwent circumcision and came from Urinary Surgery, the Affiliated Hospital of Haihan Medical College. The children＇s family members provided the informed consent, and the experiment received confirmed consent from the local ethic committee, hESCs line HN-1 was separated from human blastula. Eleven 12.5-14.5-day-old fetal mice of clean grade were selected in this study. The experimental animals were disposed according to ethical criteria. METHODS： Heads, four extremities, and viscera were removed from fetal mice under anesthesia. Subsequently, cell suspension was prepared using routine trypsinase digestion and inoculated. When cells were cultured in confluent monolayer, some primary cells were frozen, processed with mitocin-C for 2.0-3.0 hours, and inoculated based on the density of 1 × 10^8 L^-1 in gelatin-coated dish, i.e., MEF feeder layer. The HFF separation and culture and the preparation of HFF feeder layer were the same as above-mentioned processing. In addition, the two fibroblasts were respectively counted and mixed together according to the ratios of 1 ： 0, 3 ： 1, 1 ： 1, 1 ： 3, and 0：1. And then, the mixture was inoculated

关 键 词：人胚胎干细胞 成纤维细胞 饲养层 混合 

分 类 号：R394.2[医药卫生—医学遗传学]