活性氧（H2O2）信号在线虫表皮损伤修复中的功能研究

Wound healing impairment represents a major public health problem that is increasing due to an aging population and a sharp rise in the incidence of diabetes. Thus, understanding the molecular mechanisms of skin wound healing is of both biological interest and significant clinical relevance. Our long-term goal is to understand the molecular and cellular mechanisms of wound healing, using a combination of genetics and live imaging approaches. Our previous studies have established the adult C. elegans skin as a tractable genetic model system to dissect the mechanism of wound healing. We found an ancient and evolutionarily conserved role for Ca2+ as an early wound response signal in wound repair. Ca2+ signaling promotes actin cytoskeletal reorganization at wound sites by modulating the activity of Rho small GTPases. Recent results indicate that mitochondria actively responding to wounding and provide signaling to promote wound repair. This proposal specifically focuses on the roles of reactive oxygen species hydrogen peroxide (H2O2) in response to and regulating epidermal wound repair. Our preliminary data show that wounding can triggers local production of H2O2, which requires the function of NADPH dual oxidase (DUOX). Here, we propose to further investigate the molecular mechanism of H2O2 production after wounding using a combination of genetics and live imaging in C. elegans. We will then explore the role of H2O2 in triggering wound repair and its cellular and molecular mechanism. Results from these studies will not only improve our knowledge of mechanisms of wound healing, but will also shed light on the H2O2 signaling in other biological events. More importantly, a potential outcome of this research may also enable new therapeutic approaches for impaired wound healing in humans.

皮肤是人类最重要器官之一，皮肤损伤修复对人体健康具有重大医学意义。全面深入研究皮肤细胞如何响应损伤和修复伤口的细胞及分子生物学机制一直是研究人员重要的关注点。申请人在国际上率先以线虫表皮为研究对象，基于转盘式激光共聚焦显微镜活体原位时序成像技术和激光、机械损伤方法，在亚细胞和分子水平上记录表皮损伤发生和修复的全过程。申请人前期工作阐明了线虫表皮伤口愈合的分子反应过程；证实钙信号是损伤激活的最初起始信号并在伤口愈合中发挥关键作用；发现了一种由线粒体介导表皮伤口愈合的全新机制。我们将继续利用已经建立的成熟实验系统，结合显微成像技术、遗传学和分子生物学等方法研究损伤激活的H2O2信号分子在表皮损伤修复中的功能，并寻找其上游调控信号和下游靶基因，探索上下游协同作用关系。本课题将加深对表皮细胞应对损伤释放信号以及这些信号如何调控伤口修复等重要生物学过程的理解，并对人类皮肤损伤修复和再生提供重要启示。

损伤早期应激信号对后期伤口的愈合至关重要，过去的研究发现，组织损伤能快速激活钙信号，过氧化氢信号及ATP信号等。本项目主要以秀丽线虫为模型，研究损伤引起的过氧化氢信号对伤口愈合的功能和分子调控机制。在项目的资助下，课题组对这一研究内容进行了深入的分析和探索，主要取得了以下发现。首先，利用线虫活体显微成像，本项目发现损伤会迅速产生局部的过氧化氢信号，同时快速释放分散到胞质中。并且，这一过程是NADPH氧化酶所必需的。通过遗传学和伤口愈合实验，我们已经明确了H2O2信号抑制线虫表皮的伤口愈合，而降低过氧化氢信号则加速肌动蛋白重塑为基础的伤口愈合。通过药物处理，我们已经证实H2O2的抗氧化剂可以促进肌动蛋白的重塑过程。在分子机理研究方面，我们已经通过转基因敲入，单拷贝转基因插入和大量的显微成像，突变体实验发现H2O2的一个主要分子靶点是CDC-42。损伤导致CDC-42的局部聚集，类似体内相变的过程影响其活性。H2O2信号则抑制损伤引起的CDC-42的快速聚集从而抑制了伤口愈合。CDC-42的CAAX motif以及C端正电氨基酸是CDC-42快速聚集所必须的。通过基因突变，课题组发现CDC-42 105位半胱氨酸突变增强CDC-42的内源性聚集并加速伤口愈合。C105A突变导致CDC-42不能响应H2O2，影响增强其局部激活。我们推测C105可能与C188形成二硫键介导响应H2O2信号。这些发现表明在单细胞损伤过程中，CDC-42是H2O2的一个重要靶向蛋白，通过氧化还原作用和快速的相变过程调控其激活状态，为进一步理解内源性损伤应激和快速伤口愈合提供了新的理论。本项目的研究成果已经在Developmental Cell进行了评审，获得了评审专家的好评，目前我们正在努力实验回答评审意见。在项目的资助下，我们和开展了细胞膜修复的研究，并获得了重要研究成果，发表文章于Developmental Cell上。