REST调控角质形成细胞增殖/分化的平衡在创面愈合中的作用及机制研究

The balance between proliferation and differentiation of keratinocytes is a key for epidermal homeostasis and wound healing, and the cellular and molecular mechanisms still need to be explored. We detected mRNA expression profiles of human keratinocyte differentiation, the significantly reduced expression of the transcriptional repressor REST was firstly discovered and verified, and REST silencing inhibited keratinocyte proliferation, promoted differentiation and slowed wound healing. Therefore, we propose that REST can promote the regeneration of skin epidermis by regulating the balance of keratinocyte proliferation/differentiation. Based on the previous work, during this project, the expression and distribution of REST in epidermal tissue and keratinocytes will be verified; the roles of REST on the cell proliferation and differentiation will be studied through interference or overexpression; the interactions of REST, HDAC and its target genes will be investigated by the application of ChIP, EMSA and coIP, together with the screening the MAPK pathway, the regulatory mechanisms of REST on its target genes will be verified; silencing or adenovirus overexpression of REST in rat skin wound healing model will help to confirm the regulatory functions of REST on the keratinocyte proliferation/differentiation balance and its significance on wound healing in animal level. The completion of this study will be helpful to enrich the mechanisms of skin wound healing, and provide a new direction and strategy for the regulation of wound healing.

角质形成细胞增殖/分化的平衡是皮肤稳态维系和创面修复的关键，其中的细胞与分子机制尚待完善。我们检测了人角质形成细胞分化的mRNA表达谱，首次发现并验证到转录抑制因子REST表达显著下调，且沉默REST抑制角质形成细胞增殖，促进分化，减慢创面修复。因此我们提出REST通过调控角质形成细胞增殖/分化的平衡，促进皮肤表皮再生的设想。本项目将在前期工作基础上，明确REST在表皮组织及角质形成细胞中的表达和分布；通过干扰或过表达REST研究其在细胞增殖和分化中的作用；应用ChIP、EMSA和coIP研究REST与HDAC及其靶基因的互作，通过MAPK通路的筛选，明确REST对靶基因的调控机制；通过大鼠皮肤创面干扰或腺病毒过表达REST，在动物水平证实REST对角质形成细胞增殖/分化平衡的调控作用，及对创面愈合的意义。本课题的完成将有助于进一步丰富皮肤创面愈合机理，为干预创面修复提供新的方向与策略。

皮肤损伤发生后，损伤边缘的角质形成细胞加速增殖，在创面分化重建表皮。人皮肤表皮的稳态依赖于基底层和基底上层的角质形成细胞在增殖与分化之间的平衡，其中涉及的细胞与分子机制尚待完善。通过分析角质形成细胞分化的基因表达谱，发现转录抑制因子REST在细胞分化后表达降低，并在表皮组织中得到了验证。在细胞水平上，通过沉默和过表达REST研究其对细胞增殖/分化的影响，结果发现REST在维系角质形成细胞增殖中起关键作用，而REST抑制作用的解除能够促进细胞分化。转录组分析基础上进行蛋白互作研究，发现REST可能与靶基因EGR1和MAPK11互作，通过MAPK、Notch和mTOR通路调控泛素化蛋白降解，调控细胞增殖/分化的平衡。动物实验中，发现REST的异常表达影响皮肤创面愈合。综上，本项目研究发现REST通过调控角质形成细胞增殖/分化的平衡影响创面愈合，进一步丰富了角质形成细胞功能的调控机制，为干预皮肤创面愈合提供新思路。