成体表皮干细胞的分离培养与鉴定

目的 探讨成体表皮干细胞体外分离、培养和鉴定的技术方法。为组织工程皮肤的构建提供种子细胞。方法 通过中性蛋白酶消化成人包皮获得表皮细胞，再通过胰蛋白酶与乙二胺四乙酸（EDTA）消化并制成单个表皮细胞悬液，接种至人胎盘Ⅳ型胶原包被的培养瓶内．以Dulbecco改良Eagle培养基／F12（DMEM／F12，1：1）作为表皮干细胞培养基置培养箱内培养，37℃静置10～l5min，留用快速贴壁细胞继续培养，所得细胞分别以能否呈克隆状生长、流式细胞仪测细胞周期、透射电镜观察其超微结构及β1-整合素、角蛋白19（K19）免疫细胞化学染色进行鉴定。结果 人胎盘Ⅳ型胶原包被的培养瓶内快速贴壁细胞继续培养24h后细胞呈克隆状生长；电镜观察其超微结构示非成熟细胞特征；细胞周期分析示，第2代中静息期／DNA合成前期（G0／G1期）细胞占86．83％。β1-整合素、K19免疫细胞化学染色呈阳性。结论 成体表皮干细胞在体外得到成功分离与培养。

Isolation, culture and identification of adult epidermal stem cell

OBJECTIVE: To explore and establish a new method of isolation, culture, and identification of adult epidermal stem cell in vitro for the provision of seed-cells in tissue engineering of skin. METHODS: Epidermis was obtained by digesting human foreskin with protease and it was dissociated into single cells with trypsin and ethylenediaminetetraacetic acid (EDTA). These single epidermis cells were inoculated onto human collagen IV coated flasks and cultured at 37 degrees C in a humidified atmosphere containing 5% CO(2). The nonadherent cells were rinsed off 10-15 minutes after inoculation. The adherent cells were observed under phase contrast microscope and electron microscope, and they were identified with immunocytochemical methods. Cell cycles of the adherent cells were determined with flow cytometry. RESULTS: With phase contrast microscope, the rapidly adherent cells were observed to form colonies 24 hours after inoculation. Immunocytochemistry showed the rapidly adherent cells were positive for beta1-intergrin and keratin 19. Cell cycles showed that about 86.83% cells were in resting state/pre-DNA-synthetic gap (G0/G1 phase). Electron microscopy revealed that the rapidly adherent cells were immature. CONCLUSION: This study shows that adult epidermal stem cells could be isolated and cultured in vitro successfully.