Abstract: Objective To subculture the epidermal stem cells of Wuzhi Shan Pigs(WZSP) to more than 15 passages so as to establish the epidermal stem cell line of WZSP.Methods The skin pieces from the ear tissue of WZSP were digested with dispase in order to obtain the epidermis,and then the epidermis was dissociated into single cells with 0.25% tripsin and 0.02% EDTA.Then the cells were inoculated onto human collagen Ⅳ coated flasks and cultured at 37℃ in a humidified atmosphere containing 5% CO_2.The cells which didn't adhere were rinsed off 10～15 minutes after inoculation.The adherent cells were cultured in the conditional medium [including DMEM/F10(1∶1),10%conditioned medium,5 μg/mL insulin,20 ng/mL EGF,20ng/mL IGF-Ⅰand 0.5 μg/mL hydrocortisone],and subcultured with 0.25% tripsin.ESCs colonies were stained by AKP.Results With phase contrast microscope,the rapidly adherent cells were observed to form colonies 24 hours after inoculation.The ESCs have been subcultured to more than 15 passages,and the interval was about 7 days.The ESCs colonies which were stained by AKP showed red color.Conclusions It is possible to establish the ESCs line in the condition of this experiment.AKP identification demonstrates that the ESCs have the characteristics of stem cells.

Key words: WZSP, Epidermal stem cells(ESCs), Isolate, Subculture, AKP identification

摘要： 目的将五指山猪(Wuzhi Shan Pigs,WZSP)表皮干细胞传至15代以上,初步建立实验用近交系小型猪WZSP表皮干细胞系。方法采集WZSP耳组织,通过中性蛋白酶消化获得表皮细胞,再通过0.25%胰酶+0.02%EDTA消化制成单个表皮细胞悬液,接种在Ⅳ型胶原包被的培养瓶内,采用无血清条件培养基培养,0.25%胰酶消化传代。对所培养表皮干细胞进行AKP染色鉴定。结果表皮干细胞能够快速粘附在Ⅳ型胶原上,呈克隆状生长,已传至15代以上,传代间隔为7 d左右;取第5代细胞进行AKP染色呈阳性。结论在本实验条件下,建立ESCs细胞系是可能的,经鉴定,所培养细胞具有干细胞基本特性。

关键词: WZSP实验用近交系, 表皮干细胞, 分离, 传代, AKP染色鉴定