摘要
目的:探索人表皮干细胞(keratinocytestemcell,KSC)的筛选方法、体外培养条件及生长分化特性。方法:由包皮消化获得表皮细胞,以低密度单克隆筛选法挑取KSC克隆,并以挑取克隆传代及子代细胞混合传代两种不同的方法持续传代培养,采用3种不同的滋养细胞。计算每代KSC子代细胞的克隆形成率(colonyformingefficiency,CFE)以及整合素β1及外皮蛋白的表达情况。结果:将KSC克隆的子代细胞混合消化传代培养,CFE会逐渐下降,从最初的95%以上降至第6代时的25%左右。每代均可见3种处于不同分化状态的克隆混合生长,即干细胞克隆(绝大部分细胞未分化,整合素β1阳性、外皮蛋白阴性)、短暂增殖克隆(部分细胞进入终末分化)、终末分化克隆(所有细胞均发生终末分化,外皮蛋白阳性、整合素β1阴性)。以挑取克隆传代法传代培养,其CFE仍能保持在95%以上并能持续传代。结论:以低密度单克隆筛选法可成功地筛选出KSC克隆并能持续传代培养,多种成纤维细胞可作为培养KSC的滋养细胞。KSC在体外会分裂增殖并逐渐分化,但干细胞的数量比例可以保持在较高水平。
AIM:To explore th e method of isolation an d cultivation in vitro of human keratinocyte stem cells (KSCs )and characterize its proliferation and differ-entiation.METHODS:Huma n keratinocyte cells were isola ted from children' s fore-skins and cultured at a clonal densit y.Then the clone formed by KSC was isolated,cultured and subc ultured consecutively with feeder-layer of3T3cells,fetal or child' s fibrobl asts.The colony forming ef ficiency(CFE)was determined before each subculture .Meanwhile,the expression of integrinβ1and involucrin in daughter clones we re detected.RESULTS:CFE of all the daughter cells decreased from over 95%to about 25%during consecutive subculturin g for five times.Whereas CFE could be kept for more than 95%if the mega-clo ne was isolated alone from other KS C daughter cells and disaggregated,r eplated to form further clones.Thre e typ es of clones include holoclone,meroclone and paraclone co-exist in each ge n- eration of KSC daughter cells.Holoclone was formed by KSC and character-ized b y the expression of integrinβ1while mereclone and paraclone was formed by tran sit amplifying cell an d characterized by the expression of in-volucrin partly or totally.It' s a practical way to isolate KSC clone by culturing epidermal cells at a clonal density.The KSC clone can be isolated,cultur ed a nd subcultured with the feeder-layer of 3T3cells,fetal or child' s fibrob-l asts.Proliferation and different
出处
《中国临床康复》
CSCD
2004年第2期220-222,T001,共4页
Chinese Journal of Clinical Rehabilitation
基金
国家自然科学基金资助项目(30277903)~~
