TRPS1调控角质形成细胞SNAI2基因转录影响再上皮化启动的作用及机制研究

evere disability and even death could be subsequently initiated by large-area defects of skin. Therefore,to achieve a rapid re-epithelialization and complete healing is a primary goal for wound repair and regeneration researchers. Keratinocytes(Kc) acquiring mobility via epithelial-mesenchymal transition (EMT) is a committed step for priming the re-epithelialization procedure,however, the molecular machanism of EMT has not been fully understood.A DNA microarray analysis of acute and chronic wound epidermis was performed and a series of genes with variable transcription activity were screened out, incluing TRPS1 and SNAI2,which are both pivotal transcriptional regulator involed in EMT. Further research found that with the promotion of TRPS1 in Kc cultured in vitro,SNAI2 expression induced by TGF-β1 could be dramatically inhibited. In combination of whose nature as a transcriptional repressor and analysis of bioinformation softwares, we presume that SNAI2 could be the down-stream target gene that directly repressed by TRPS1,hence EMT in Kc would be negatively regulated, which ultimately resulting in a retardation of re-epithelialization. In order to confirm this presumption, we anticipate to apply realtime fluorescence quantitative PCR, transwell, chromatin immunoprecipitation(ChIP) and luciferase reporter gene assay et cetera to explore the impacts of TRPS1 on Kc EMT , and the potential signal pathway through which SNAI2 transcription being regulated ,by means of enhancing or inhibiting TRPS1 expression both in vivo and in vitro. This research will be in great help to further understand the molecular mechanism of re-epithelialization, and to provide a new sight of view guiding the clinicians to find a more efficient strategy for treating skin wounds.

皮肤大面积缺损可致严重残疾甚至死亡，促进创面愈合是创伤修复研究的重要目标。现认为，角质形成细胞（Kc）向间质细胞转化获得移行能力是启动再上皮化、修复创面的关键。前期基因芯片检测显示在急/慢性创面组织中TRPS1及SNAI2基因存在差异表达，两者是调控上皮-间质转化（EMT）的重要转录因子，体外研究显示增强TRPS1表达能显著抑制SNAI2表达；结合有关文献推测：TRPS1在创面修复过程中发挥了重要作用，其机制与直接抑制SNAI2转录后负向调控Kc EMT有关。本研究拟在构建TRPS1过表达/干扰载体的基础上，通过荧光定量PCR、WB、ChIP、荧光素酶检测等技术在细胞水平阐明TRPS1对Kc间质化的影响及其调节SNAI2表达的信号转导机制，同时在动物水平验证TRPS1调控Kc移行在创面愈合中的作用。研究结果有望从EMT角度阐释再上皮化的调控机制，为临床寻找有效促愈方法提供理论依据。

皮肤创面愈合机制一直是创伤修复领域研究的重点。再上皮化是皮肤创伤愈合过程中的重要事件，是一个以角质形成细胞（Kc）为主导的多因素协同作用的过程，Kc交叠发生移行、增殖、分化是实现再上皮化的细胞生物学基础，其中细胞发生移行，是再上皮化的启动环节及限速步骤；而发生于 Kc移行过程中的一系列细胞功能和标志物的改变，实质是 Kc的上皮-间质转化（EMT）。前期基因芯片检测显示在急/慢性创面组织中 TRPS1 及SNAI2 基因存在差异表达，是调控EMT的重要转录因子，增强 TRPS1 表达能显著抑制 SNAI2 表达，结合有关文献推测：TRPS1 是KcEMT的关键负向调控因子，其表达上调能抑制 Kc EMT；SNAI2 是受 TRPS1 直接调控的下游基因，TRPS1 通过与 SNAI2 的启动序列结合，抑制其转录，负向调控 KcEMT。为证实上述调控机制，本研究首先在细胞水平探讨 TRPS1 在创面再上皮化过程中的作用，构建 TRPS1过表达及干扰载体感染体外培养Kc细胞，Transwell 检测发现TRPS1过表达组Kc细胞迁移能力显著下降，RT-PCR 、WB等技术检测 KcEMT 标志物 Vimentin、E-Cadherin变化受阻，SNAI2表达显著降低；进一步构建 SNAI2 基因过表达及干扰载体慢病毒载体，转染Kc细胞并筛选出稳定表达克隆，继而以 TRPS1干扰或过表达病毒载体感染细胞，检测 Kc 细胞移行能力及 EMT 相关指标改变，明确 SNAI2是TRPS1调控创面再上皮化中的下游介导分子；再克隆 SNAI2 基因启动子，构建截短型启动子，采用荧光素酶报告系统明确启动子核心活性区域，利用生物信息学对 TRPS1 潜在结合位点预测分析，定点突变技术构建突变体启动子并测定活性，染色质免疫共沉淀及凝胶电泳迁移阻滞技术明确 TRPS1 在SNAI2 基因启动子上的结合位点；最后利用 SKH-1无毛小鼠构建创伤模型，在动态观察创面愈合过程中 TRPS1、SNAI2 表达及 Kc EMT 标志物的变化的基础上，施以 TRPS1及 SNAI2 相关病毒分子处理创面，阐明TRPS1通过SNAI2的介导对创面愈合的负向调节作用。研究明确了 TRPS1通过直接抑制 SNAI2 转录负向调控 Kc EMT的关键作用，为加速再上皮化进程、促进创面愈合提供了新思路。