人表皮干细胞的体外分离和培养

背景：如何获得高纯度的表皮干细胞以及维持其干细胞表型和体外增殖特性，是目前表皮干细胞体外培养过程中急需解决的问题。目的：建立人表皮干细胞体外分离和培养的技术方法。设计、时间及地点：细胞观察，于2005-03／2006-05在南昌大学第一附属医院烧伤科完成。材料：所用包皮来自2～12岁行包皮环切术的患者，患者无泌尿系统感染等疾病。方法：取包皮皮片，采用DispaseⅡ酶和胰蛋白酶两步法分离出角朊细胞和成纤维细胞，Ⅳ型胶原快速黏附法富集分离表皮干细胞。收集处于指数生长期第二三代成纤维细胞的上清液，过滤后与角朊细胞无血清培养基等比例混合，并加入胎牛血清、表皮生长因子、牛垂体提取物等组成表皮干细胞培养液，用以人表皮干细胞的体外扩增培养，以角朊细胞作对照。主要观察指标：传至第2代，通过平板克隆形成实验计算克隆形成率和克隆维持时间，采用免疫组织化学染色检测β1整合素和角蛋白19的表达。结果：①与同一标本同一批次培养的角朊细胞相比，人表皮干细胞的克隆形成率明显升高，克隆维持时间延长（P〈0．01）。②表皮干细胞β1整合素及角蛋白19免疫组织化学染色均呈阳性。结论：应用Ⅳ型胶原快速黏附法及人成纤维细胞条件培养基，对人表皮干细胞成功进行了体外分离和培养。

In vitro isolation and culture of human epidermal stem cells

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.