目的探讨表皮干细胞(epidermal stem cells,ESCs)诱导分化为汗腺细胞(sweat gland cells,SGCs)过程中表型的改变及其通路的调控机制。方法取健康成人包皮,采用中性蛋白酶消化后高浓度Ⅳ型胶原黏附法分离培养获得ESCs,行β_1整合素、角蛋白19(cytokeratin 19,CK19)及p63免疫荧光染色法鉴定;取健康成人全层皮肤,采用Ⅱ型胶原酶消化法分离提取汗腺组织,体外增殖培养SGCs,行CK7、CK18、CK19和癌胚抗原(carcinoembryonic antigen,CEA)免疫荧光法鉴定。取第2代ESCs分为4组:A组将ESCs及SGCs两种细胞通过Ttranswell共培养系统共培养,B组通过单纯添加汗腺上清液培养ESCs,C组在A组基础上加入浓度为60 ng/mL的EGF,D组在A组基础上加入浓度为10 mmol/L的PD98059。分别通过倒置相差显微镜进行细胞形态学观察、流式细胞术检测ESCs表型阳性率及Western blot检测分化过程中的信号通路机制。结果经细胞形态学观察及免疫荧光染色鉴定提示培养细胞为ESCs和SGCs。倒置相差显微镜观察示,A、C、D组培养后细胞形态变化相似,9 d后细胞开始出现扁平多角形结构改变;B组形态变化较慢培养12 d后与其他3组结构相似。流式细胞术结果示,与B组比较,A组Transwell共培养系统共培养后,ESCs的β_1整合素阳性率显著下调、CEA阳性率显著上调(P<0.05);C组加入EGF可降低共培养系统中ESCs的β_1整合素下调和CEA上调,D组加入PD98059可增强共培养系统中ESCs的β_1整合素下调和CEA上调,A、C、D组间比较差异均有统计学意义(P<0.05)。Western blot检测示,4组均有较高水平细胞外信号调节激酶(extracellular signal regulated kinase,ERK)表达,但B组明显低于其他3组(P<0.05);磷酸化-ERK表达A组最高、C组最低,各组间比较差异均有统计学意义(P<0.05)。结论处于SGCs生长环境中时,通过Transwell共培养系统共培养,ESCs可经诱导分化为SGCs,其表型发生相应改变;通过对丝裂原活化蛋白激酶/ERK通路的控制,可以调节ESCs的分化方向和程度。 Objective To investigate the phenotypic changes and the regulatory mechanism of epidermal stem cells (ESCs) during their differentiation into sweat gland cells (SGCs). Methods The preputial adult foreskin was harvested for immunohistochemistry with β 1 integrin, cytokeratin 19 (CK19) and p63 immunofluorescent staining after neutral protease digestion and high concentration type Ⅳ collagen adhesion. Layer of skin, type Ⅱ collagenase digestion was used to extract the sweat gland tissue, in vitro proliferation of cultured SGCs, CK7, CK18, CK19 and carcinoembryonic antigen (CEA) immunofluorescence identification. The second generation of ESCs were divided into four groups: group A, ESCs and SGCs were co-cultured with Ttranswell co-culture system, group B was cultured with simple addition of sweat gland supernatant, group C was added to group A with a concentration of 60 ng / mL of EGF, group D was added PD98059 at a concentration of 10 mmol / L on the basis of group A. The morphological changes of the cells were observed by inverted phase contrast microscope. The positive rate of ESCs was detected by flow cytometry and the signal transduction mechanism was analyzed by Western blot. Results The cell morphology and immunofluorescence staining showed that the cultured cells were ESCs and SGCs. Inverted phase contrast microscopy showed that the morphological changes of cells in groups A, C and D were similar. After 9 days, the cells began to show flat polygonal structure. The morphological changes of group B were slower than those of the other three groups after 12 days of culture. Flow cytometry showed that compared with group B, the positive rate of β 1 integrin in ESCs was significantly decreased and the positive rate of CEA was significantly increased (P <0.05) after co-cultured with Transwell co-culture system in group A The down regulation of β_1 integrin and the upregulation of CEA of ESCs in culture system and the addition of PD98059 in group D enhanced the downregulation of β_1 integrin and the upregulation of CEA in ESCs in co-culture system, with significant differences between A, C and D groups (P < 0.05). Western blot showed that the expression of extracellular signal regulated kinase (ERK) was higher in all four groups, but the expression of ERK in group B was significantly lower than that in the other three groups (P <0.05); the phosphorylation-ERK expression in group A was the highest , The lowest in C group, the difference between the two groups was statistically significant (P <0.05). Conclusions When cultured in the environment of SGCs, the ESCs can be induced to differentiate into SGCs by co-culture of Transwell co-culture system. The phenotype of ESCs is changed accordingly. The differentiation of ESCs can be regulated by the control of mitogen-activated protein kinase / ERK pathway Direction and degree.