热损伤与碱性成纤维细胞生长因子联合处理诱导表皮细胞去分化的实验研究

目的：体外建立表皮细胞去分化模型，为深入阐明表皮细胞去分化过程奠定基础。方法：离体条件下选择热损伤与碱性成纤维细胞生长因子（bFGF）作为联合诱导因素处理成熟表皮细胞。通过Giemsa染色和电镜观察细胞形态的改变；通过细胞培养和滋养层培养体系观察细胞的增殖能力，包括克隆形成和复层生长情况；采用器官培养体系体外构建三维皮肤，观察细胞再分化形成表皮情况；应用基因芯片技术观察细胞基因表达的改变。结果：经联合诱导后，细胞形态发生改变，表现为体积减小，细胞器数目减少，核浆比增大。增殖和再分化能力的改变体现在诱导后的表皮细胞能形成克隆，在滋养层培养条件下可以形成复层生长，并能够在体外构建出包括基底层和基底上层在内的表皮结构。基因水平的改变表现为与角质化相关基因下调和与有丝分裂相关基因上调。结论：在热损伤与bFGF联合诱导下，已分化的表皮细胞会发生形态、功能和基因表达的改变，表现为干细胞的特性。

Dedifferentiation of human epidermal keratinocytes induced by combined heat and basic fibroblast growth factor treatment

Objective：To reproduce a dedifferentiation of epidermal cells model in vitro for the purpose of clarifying the process of dedifferentiation of epidermal cells. Methods： Mature epidermal cells were exposed to heat and basic fibroblast growth factor （bFGF） treatment in vitro. Giemsa staining and electron microscopic analysis were performed to observe the morphological change; the proliferation capacity was assessed by two-and feeder-layer culture systems and the redifferentiation potential by organotypic cultures; gene chip technology was utilized to analyze the differences in gene expression profile before and after induction. Results： After exposure to heat and bFGF treatment, cells reverted from a differentiated to a dedifferentiated state. They showed morphological changes including reduction in size and reduction in number of cellular organeltes. They also regained a strong proliferation capacity,and the skin equivalent constructed with these cells showed a well-organized structure, with keratin 14 （CK14）, CK19 and β1 integrin-positive cells in the basal layer. For the gene expression profile, genes controlling cell adhesion and mitotic cell cycle were upregulated during the reversal process, but those controlling cell differentiation and keratinization, were downregulated. Conclusion： Heat and bFGF could induce a reversial of epidermal cells from an adult-differentiated state to an immature-like dedifferentiated state as evidenced by their changes in morphology, function and gene expression.