生理强度电场抑制E-cadherin表达促使表皮细胞低分化的作用与机制研究

The maintenance of low differentiation is an important feature of neonatal epidermis, and acts as the key to determine the migratory phenotype and motility of keratinocytes during wound reepithelialization. We found in vivo for the first time that wound endogenous electric field is an important signal to inhibit epidermal differentiation, but the mechanism is not clear. Based on a critical role for E-cadherin in mediating epidermal differentiation that has been well identified previously and our current findings, we here proposes a hypothesis that electric field signal inhibits E-cadherin transcription via the EGFR/ERK/Snail pathway, thereby inhibiting keratinocyte differentiation. We firstly intend to further confirm a role of electric field in keratinocyte differentiation in vivo and in vitro, and then elucidate that the suppressed E-cadherin and its mediated differentiation pathways accounts for the low differentiation of keratinocyte induced by electric field. Using techniques such as gene silencing et al., we plan to reveal that EGFR/ERK activation and up-regulation of transcription inhibitory activity of Snail are the key signal mechanisms for E-cadherion downregulation, and further check out how ERK regulates Snail from its transcription to intracellular distribution, and which molecular downstream of ERK responsible for the direct interaction with Snail. Finally, by using EGFR mutant mice, we intend to verify the signal mechanisms responsible for wound endogenous electric field suppressed epidermal differentiation in vivo. Therefore, this study would be of great significance in revealing a new mechanism of epidermal differentiation regulation, innovating the new theory for electric field promoting reepithelialization, and deepening our understanding on wound healing and even unhealing mechanisms.

低分化状态的维持是创缘新生表皮的重要特征，是决定表皮细胞移行表型和运动性的关键。我们首次发现，创缘电场具有抑制表皮分化的重要作用，但机制不清楚。基于E-cadherin介导表皮分化的认识，结合工作基础，本研究提出电场信号经由EGFR/ERK/Snail途径，抑制E-cadherin转录，从而抑制表皮分化的假设，拟首先从体内外水平进一步明确电场抑制表皮细胞分化的作用，继而阐明E-cadherin表达及其介导的分化途径受抑是其重要机制；采用基因沉默等技术，揭示EGFR/ERK激活、上调Snail的转录抑制活性是E-cadherin表达受抑的关键；从表达到分布，探寻ERK调节Snail的可能环节与分子基础；最后，利用EGFR突变体转基因小鼠在体验证创缘电场抑制表皮分化的作用机制和意义。研究对揭示创面表皮分化调节新机制，创新电场促上皮化作用新理论，深化创面愈合乃至创面难愈机制的认识，具有重要意义。

低分化状态的维持是创缘新生表皮的重要特征，是决定表皮细胞移行表型和运动性的关键。我们前期发现，创缘电场具有抑制新生表皮分化的重要作用，但作用机制不清楚。本研究提出“电场经由EGFR/ERK/Snail途径，抑制E-cadherin转录，从而抑制表皮分化”的假设并予以验证。研究首先以体外培养的表皮细胞单层为对象，发现在生理强度电场作用下，表皮细胞间粘附分子-E-cadherin和表皮细胞分化标志K1和K10的表达均显著下调；通过基因调控实验，发现E-cadherin的表达水平与表皮细胞单层的分化状态显著相关，提示细胞间粘附可能是调节表皮细胞单层分化状态的重要因素。对稳定高表达E-cadherin的表皮细胞单层施加生理强度电场，发现生理强度电场能部分逆转E-cadherin高表达及其诱导的表皮细胞分化，揭示生理强度电场通过降低细胞间粘附进而抑制表皮细胞单层分化的重要作用。进一步，通过转录组测序，我们筛选出电场作用条件下抑制E-cadherin表达的可能转录因子-Snail；通过基因干扰实验证实，Snail是生理强度电场作用条件下抑制E-cadherin表达的关键转录调节因子。对Snail上游信号的研究发现，尽管生理强度电场显著激活上游ERK信号，但该信号不参与对Snail表达的调节。通过转录组测序、KEGG富集分析和抑制剂抑制实验，我们发现PI-3K/AKT是介导生理强度电场增强Snail转录活性、进而抑制E-caderin表达和表皮细胞分化的关键上游信号机制。最后，我们建立巴马香猪皮肤全层缺损创面模型，通过对创面施加反向电场削减创面内源性电场，发现创面再上皮化延迟、新生表皮呈现出明显的细胞分层和形成更多的钉突等高分化表型，同时新生表皮细胞间E-cadhersin的表达以及新生表皮分化标志物表达显著升高，体内证实创缘电场通过抑制表皮细胞间粘附分子-E-cadherin的表达进而使新生表皮维持低分化状态。研究对揭示创面表皮分化调节新机制，创新电场促上皮化作用新理论，深化创面愈合乃至创面难愈机制的认识，具有重要意义。