小鼠胚胎及人包皮成纤维细胞按比例制成混合饲养层上的人胚胎干细胞生长状态  

Growth state of human embryonic stem cells on mixed feeder layers with mouse embryonic fibroblasts and human foreskin fibroblasts at different ratios

作　　者：李斌[1,2] 彭秋平[3] 卢伟英[1,2] 徐雯[1,2] 金应霞[3] 黄元华[1,2] 

机构地区：[1]海南医学院 [2]海南医学院附属医院生殖医学中心,海南省海口市570102 [3]海南医学院附属医院生殖医学中心

出　　处：《中国组织工程研究与临床康复》2008年第3期424-428,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：目的: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。欲解决以小鼠胚胎成纤维细胞或人包皮成纤维细胞为常规饲养层培养人胚胎干细胞时存在的问题,观察两者按一定比例制成的混合饲养层上人胚胎干细胞的生长状态。方法: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。①对象: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12.5～14.5d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。②实验方法:将去除头、四肢和内脏的胎鼠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1×108L-1密度接种于明胶包被的中心皿内,即小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1×108L-1密度接种于明胶包被的中心皿内,即混合饲养层。③实验评估: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。结果:①不同饲养层上人胚胎干细胞的生长状态比较:生长在小鼠胚胎成纤维细胞和人包皮成纤维细胞上的人胚胎干细胞克隆扁平、不饱满,而生长在混合饲养层上的人胚胎干细胞克隆饱满、厚实,其克隆形态显著好于其他两种饲养层。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:小鼠胚胎成纤维细胞AIM： The key of the human embryonic stem cell culture is to guarantee the totipotency and inhibit spontaneous differentiation. There are the problems in the traditional methods that human embryonic stem cells were cultured on the mouse embryonic fibroblasts or human foreskin fibroblasts as feeder layer. We aimed to solve the problems, observe growth state of human embryonic stem cells when it was cultured on the mixed feeder layers of different proportion. METHODS： Experiments were completed in Reproductive Medical Center of Hainan Medical College from April 2006 to July 2007. （1）The foreskin was from the boy who was circumcised, provided by Department of Urinary Surgery of Hospital Affiliated to Hainan Medical College. Child guardian signed an informed consent of the treatment and experiment and the experiment was approved by the hospital medical ethics committee. Human embryonic stem cell line （HN-1） was isolated from human blastocysts and identified. Eleven clean fetal mice of 12.5-14.5 d were collected and the disposal of animal conformed to the animal ethics standards during the experiments. （2）The fetal mice whose heads, limbs and internal organs were removed were repeatedly digested by the trypsin to obtain cells, then cells were cultured. Parts of the original cells were cryopreserved after confluence. It was the mouse embryonic fibroblast feeder layer that cells which were treated for 2.0-3.0 h with mitomycin C were cultured in the center plate coated by gelatin at 1 ×10^8 L^-1. Isolation, culture and preparation of feeder layer of human foreskin fibroblast were the same as above. Mixed feeder layer was that cells which were mixed according to 1 ： 0, 3 ; 1, 1 ： 1, 1 ： 3, 0 ： 1 were cultured in the center plate coated by gelatin at 1×10^8 L^-1. （3）Growth states of human embryonic stem cells were observed in three different feeder layers and undifferentiated phenotypes were detected, including expression of alkaline phosphatase, and presence of OCT-4, Tert and cell marker （O

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]