体外诱导表皮细胞去分化的初步实验研究

Primary studies on induced dedifferentiation of epidermal cells in vitro

目的建立表皮细胞去分化体外模型,探索与去分化过程有关的信号通路。方法分离培养人表皮细胞,分别用双氧水、ERK阻断剂PD98059加双氧水处理细胞,采用免疫细胞化学染色法和Western blot法检测不同方式处理后表皮细胞表型的变化情况,Western blot法检测双氧水处理后ERK信号通路相关蛋白的表达情况。结果双氧水处理后,表皮细胞干细胞标志物(CK19、β1整合素、Oct4和p63)表达增加,分化细胞标记物(CK10)表达减少,ERK信号通路中相关的磷酸化蛋白表达增强。以PD98059阻断ERK通路的活化后,双氧水诱导的表皮细胞表型逆转受到了抑制。结论ERK通路参与了双氧水诱导的表皮细胞去分化过程。

Objective To induce dedifferentiation of epidermal cells in vitro, and to explore the possible signaling pathway involved in this reversion process. Methods Differentiating epidermal cells were isolated and cultured in vitro, then were treated with H2O2. Immunocytochemistry and Western blot techniques were used to observe the phenotypical changes of these epidermal cells including the re-expression of CK19, 1 integrin, p63 and Oct4 after they had been treated with H2O2. Components of ERK MAPK signaling pathway, including p-raf, p-MEK1/2, t-MEK1/2, p-ERK1/2, tERK1/2 and c-myc, were respectively examined in the reversion process induced by H2O2. The phenotypical changes were also observed after inhibition of ERK kinase activities with specific inhibitor PD98059. Results The differentiated cells were positive for differentiated cell marker CK10 and negative for stem cell markers. The expressions of stem cell markers including CK19, 1 integrin, p63 and Oct4 increased at different levels after the epidermal cells were treated with H2O2, whereas the expression of CK10 decreased. The expressions of pertaining proteins of ERK signaling pathway were also increased during the process. After ERK pathway was inhibited by PD98059, the phenotypical reversion induced by H2O2 was suppressed. Conclusion These data collectively provide a proof-of-concept that H2O2 treatment on HENs is capable of inducing a phenotype reversion from an adult differentiated state to an immature-like dedifferentiated state via ERK MAPK-dependent pathway. It may offer the direct evidence for the existence of dedifferentiation and the underlying mechanisms involved in the process, which may bring a new insight for the regenerative medicine.