目的：诱导人胚胎干细胞（human embryonic stem cells，hESCs）定向分化为角质形成细胞K-hESCs（keratinocyte derived from human embryonic stem cells）， 并分析诱导过程中K-hESCs不同标志物的表达特点。方法：运用含骨形成蛋白4、维甲酸和N2添加剂的上皮分化培养基直接诱导hESCs分化为K-hESCs，通过染色体核型分析检测K-hESCs核型，通过实时定量PCR检测K-hESCs在分化的不同时期基因的表达及其与原代人牙龈上皮细胞（human gingival epithelial cells，HGECs）、人永生化口腔上皮细胞（human immortalized oral epithelial cells，HIOECs）、人永生化皮肤角质形成细胞HaCaT的基因表达差异；利用免疫组织化学检测不同标志物在K-hESCs的蛋白表达。结果：运用上皮分化培养基成功地诱导hESCs分化为上皮样细胞K-hESCs； K-hESCs核型具有正常46条染色体，无结构异常；K-hESCs中角质形成细胞标志物基因p63的表达明显低于HaCaT细胞（P<0.05），而与HGECs和HIOECs中的表达差异无统计学意义（P>0.05）。结论：运用上皮分化培养基可成功诱导hESCs分化为具有正常核型的K-hESCs；标志物的表达特点提示诱导hESCs分化为K-hESCs的过程是从hESCs向单层上皮干细胞分化继而转向复层上皮终末分化发展的趋势，最终得到的K-hESCs类似于单层鳞状上皮干细胞向终末分化初始阶段的角质形成细胞，该阶段的细胞由上皮干细胞静止状态被激活，处于分化初始高增殖活力阶段。
Objective: To differentiate human embryonic stem cells (hESCs) into keratinocytes (K-hESCs) and analyse the expression characteristics of biomarkers of K-hESCs. Methods: The hESCs of line H9 were seeded on matrigel in mTeSR1 medium. The hESCs were directly differentiated into keratinocytes in epithelial differentiation medium with bone morphogenetic protein 4, retinoic acid and N2 supplement. The karyotype of K-hESCs was analyzed, comparing the gene expression differences of K-hESCs with human gingival epithelial cells (HGECs), human immortalized oral epithelial cells (HIOECs) and HaCaT by Realtime PCR. Molecular characteristics of the cell differentiation were observed throughout the process by immunocytochemical techniques. Results: H9hESCs were successfully differentiated into the cells that exhibited characteristics of keratinocytes in epithelial differentiation medium. The karyotype of K-hESCs was 46, XX; and the keratinocyte gene p63 expression in K-hESCs was significantly lower than that in HaCaT (P<0.05), but there was no significant difference of p63 expression in K-hESCs, comparing with that in HGECs and HIOECs (P>0.05). Conclusion: H9-hESCs could be directly differentiated into K-hESCs. The gene expression of K-hESCs was similar to that of epithelial cells in the early stage of monolayer cells differentiation with high proliferative activity.
