人表皮干细胞的体外培养与鉴定  

In vitro isolating culture and identification of human epidermal stem cells

作　　者：江惟苏[1] 李镜波[1] 魏代奎[1] 黎丽芬[1] 胡萌[1] 戴新民[1] 

机构地区：[1]中山大学附属东华医院皮肤科,广东东莞523110

出　　处：《中国现代医学杂志》2009年第17期2683-2686,共4页China Journal of Modern Medicine

基　　金：广东省医学科学技术研究基金(No:A2006751);东莞市科技计划项目(No:200680)

摘　　要：目的探讨人表皮干细胞的体外分离培养方法,并对培养的表皮干细胞进行鉴定。方法运用中性蛋白水解酶和胰蛋白酶两步法分离表皮层和真皮层,并获得表皮单细胞悬液,采用Ⅳ型胶原铺板选择性黏附分离角质形成细胞,应用表皮干细胞培养基进行培养。倒置显微镜下观察培养细胞的生长状况,免疫组织化学染色观察表皮干细胞β1整合素、角蛋白19(K19)、CK10的表达。结果组织学观察显示,培养中实验组细胞呈现克隆生长,且克隆维持时间较长;免疫组织化学染色显示,实验组的培养细胞β1整合素及K19均呈阳性表达,而CK10阴性表达。结论应用Ⅳ型胶原黏附和运用表皮干细胞培养基可以实现人体表皮干细胞的分离和培养,为表皮干细胞体外的大量扩增奠定了基础。[Objective] To explore a method for isolating culture of human pidermal stem cells in vitro and identify human pidermal stem cells. [ Methods ] Epdermis and single epidermis cells was obtained by digesting human for skin with Dispase Ⅱ and Trypsin, then suspension of single epidermis cells on the epidermal stem cell medium （ESCM）. These single epidermis cells were inoculated on human collagen Ⅳ coated flasks and cultured on the ESCM. The cell growth was observed through inverted microscope. Immunoeytoehemistry was used to observe the expression of 131 integrin, keratin 19 （CK19）, CKI0 on pidermal stem cells. [Results] It was revealed by histological observation that colonies of the isolated and cultured cells were formed after inoculation, the time of clone sustain was longer. Positive expression of 131 integrin and K19 of the most cultured cells was detected by immunocytochemistry and CK10 was negative in the experimental group. [ Conclusion] Epidermal stem cells could be successfully isolated and cultured by adhension with type Ⅳ collagen and culture with ESCM.

关 键 词：表皮干细胞 细胞分离 细胞培养 鉴定 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]