EGF/EGFR定向诱导人表皮干细胞向汗腺样上皮分化的作用及机制研究

Epidermal stem cells(ESCs) are a kind of tissue specificity and multilineage defferentiation stem cells that can differentiate into sweat glands, hair follicles and epidermis. However, the mechanism of directed differentiation is not clear. In our previous study, cultured human ESCs which were seeded into tissue-engineering skin, could be formed into gland-like structures under particular concentration of EGF. Comparing the morphologhy, histology and biological function of induced structures and the normal sweat glands, we could conclude that there were similarities between the two. Combined with our previous study and related literature, we propose: EGF/EGFR activates PI3K/AKT, induces the EDa gene's expression, which were involved in the ESCs' differentiation into glandular epithelium-like cells. To further clarify the hypothesis, this study will deploy tissue engineering technology, morphological observation, RT-PCR, WesternBlot, vector construciton to survey the effect of EGF/EGFR in human ESCs' directed differentiation, to investigate whether the PI3K/AKT pathway by inducing EDa gene's expression is involved in the directed differentiation process. This study will elucidate the mechanism of EGF/EGFR in the ESCs'differentiating into glandular epithelium-like cells, reveal the important significance of ESCs' directed differnetiation, and also provide reliable experimental evidence of tissue-engineering skin with sweat glands.

表皮干细胞是皮肤组织特异性、有多向分化潜能的细胞，具有向汗腺、皮脂腺及毛囊分化的能力，但其定向诱导分化的机制尚不明确。课题组研究发现，将人表皮干细胞接种至复方壳多糖组织工程皮肤，一定浓度的EGF能够诱导其形成管腔样结构，该结构在形态学、组织学及功能上与在体汗腺有一定相似性。结合前期研究及相关文献，我们提出："EGF/EGFR激活PI3K/AKT，诱导EDa基因表达，参与表皮干细胞向汗腺样上皮分化"的假设。为验证该假设，本项目拟采用组织工程技术、形态学观察、RT-PCR、WB、载体构建等方法，研究EGF/EGFR在表皮干细胞定向诱导分化中的作用，探讨PI3K/AKT信号通路是否通过诱导EDa基因表达，参与该定向诱导分化过程。该研究对阐明EGF/EGFR在人表皮干细胞向汗腺样上皮分化中的机制、揭示表皮干细胞的定向分化具有重要意义，也为研制带汗腺的人工皮肤以及为创面的功能性愈合提供新的思路和策略

课题组研究发现，将人表皮干细胞接种至复方壳多糖组织工程皮肤，一定浓度的EGF（浓度为 20ng/ml），垂直震荡培养，能够诱导表皮干细胞向汗腺样上皮分化。EGF作用于复方壳多糖组织工程皮肤，EGFR的表达有明显升高。使用一定浓度的EGFR抑制剂（25mM）阻断EGFR表达，通过RT-PCR、WB、形态学观察等方法发现：EGFR及下游的PI3K、AKT、EDA基因/蛋白表达量降低，表皮干细胞的分化受限，在相同浓度的EGF作用下不能形成管腔样结构。使用一定浓度的及PI3K抑制剂（0.4uM）阻断PI3K的表达，通过RT-PCR、WB、形态学观察等方法发现：PI3K、AKT、EDA基因/蛋白表达量降低，表皮干细胞的分化受限，在相同浓度的EGF作用下不能形成管腔样结构。实验说明了EGF通过EGFR使表皮干细胞中PI3K/AKT信号通路激活，作用于EDa基因，形成管腔样结构，诱导表皮干细胞向汗腺样上皮的分化，阐明EGF/EGFR在诱导人表皮干细胞向汗腺上皮分化中的重要作用及分子作用机制。目前该课题获得国家发明专利授权2项，投稿SCI文章2篇。