中国组织工程研究与临床康复2008，Vol.12Issue(43)：8597-8600,4.
人表皮干细胞的体外分离和培养
In vitro isolation and culture of human epidermal stem cells
曾元临 1辛国华 1邱泽亮2
作者信息
- 1. 南昌大学第一附属医院烧伤科,江西省南昌市,330006
- 2. 丽水市人民医院ICU,浙江省丽水市,323000
- 折叠
摘要
Abstract
BACKGROUND: How to acquire high-purity epidermal stem cells (ESCs) and maintain ESC phenotype and in vitro proliferation characteristics are urgently solved problems in the in vitro culture of ESCs.OBJECTIVE: To establish methods to in vitro isolate and culture human ESCs.DESIGN,TIME AND SETTING: Cell observation was performed in the Department of Bum,First Affiliated Hospital of Nanchang University.MATERIALS: Foreskin was obtained front 2-12-year-old patients who were subjected to circumcision.These patients did not suffer from urinary system infection.METHODS: Keratinocytes and fibroblasts were isolated from foreskin using Dispase Ⅱ and trypsin.ESCs were isolated by rapidly adhering to type Ⅳ collagen.The supematant fluid of fibroblasts of passages 2-3,which were in the exponential growth phase,was collected.After screening,the supematant fluid was mixed with serum-free medium of keratinocytes at the proportion of 1:1.Simultaneously,fetal bovine serum,epidermal growth factor,and bovine pituitary extract were added to prepare medium of ESCs,which was used for in vitro proliferation of human ESCs.Homologous keratinocytes were used as controls.MAIN OUTCOME MEASURES: After 2 passages,cloning efficiency and cloning sustaining time were calculated by plate clone formation assay.Expression levels of β 1 integrin and keratin 19 were detected by immunohistochemistry.RESULTS: The cloning efficiency of human ESCs was significantly increased,and the cloning sustaining time was significantly prolonged as compared with homologous keratinocytes cultured in the same batch (P ＜ 0.01).lmmunohistochemistry results demonstrated that both β 1 integrin and keratin 19 were positive in the human ESCs.CONCLUSION: Keratinocytes could be successfully isolated by means of rapid adherence to type Ⅳ collagen and cultured with conditioned medium in vitro.关键词
表皮干细胞/体外/分离/培养分类
医药卫生引用本文复制引用
曾元临,辛国华,邱泽亮..人表皮干细胞的体外分离和培养[J].中国组织工程研究与临床康复,2008,12(43):8597-8600,4.
