晚期氧化蛋白产物通过mTOR通路调节角质形成细胞凋亡过程中自噬反应的研究

Diabetic chronic wounds have brought heavy load to the patients. Laboratory examinations have detected in these patients the accumulation of many oxidative products, such as advanced oxidation protein products (AOPPs), and they directly participate in the damage that oxidative stress causes which become a pivotal role in the diabetic chronic wounds. Keratinocytes are essential for re-epithelialization during wound healing process. According to our recent study, AOPPs could induce human keratinocytes apoptosis, and we accidently detected an increased expression of LC3-II, a landmark of autophagy. As a complex regulator and closed companion of apoptosis, autophagy has been a hot spot of research for decades. mTOR, a signal integrator that senses stress conditions such as oxidative products accumulation, mediates autophagy initiation, and overload damaged proteins and organelles can be degraded through autophagy process to attenuate oxidative damage to the cell. However, the role and mechanism of autophagy in AOPPs induced human keratinocytes apoptosis have not been fully elucidated. Our purpose is to find our whether AOPPs can affect keratinocytes autophagy via mTOR pathway during their way of inducing cell apoptosis. In this study, in vivo and in vitro experiments were conducted on human epidermis, obtained from diabetic wound margin, and human immortalized keratinocyte (HaCaT) cells, substitute of human keratinocytes, to examine the effects of AOPPs on keratinocytes autophagy and the underlying cellular mechanism. Hopefully, our study could provide a new thought of revealing the mechanism of keratinocytes overcoming oxidative stress during wounds healing process.

糖尿病慢性创面严重影响患者的生活质量，在这些患者体内，晚期氧化蛋白产物（AOPPs）等氧化物水平明显升高，直接引起氧化应激水平上调，成为创面愈合延迟的始动因素之一。角质形成细胞是创面表皮再生的主要参与者，本课题组前期研究发现，AOPPs可诱导角质形成细胞凋亡增加，并且观察到自噬相关蛋白LC3-II的表达增加。作为细胞凋亡的调控因素之一，细胞自噬是近年来的研究热点。氧化产物刺激下，细胞可通过mTOR通路调节自噬反应水平，减轻氧化应激对细胞的损伤，但在AOPPs诱导角质形成细胞凋亡中自噬的调节通路尚未明确。本研究旨在探究AOPPs能否通过mTOR自噬反应通路参与角质形成细胞凋亡的调节。本项目以糖尿病慢性创面边缘表皮组织及HaCaT细胞为对象，从体内、体外层面探讨AOPPs通过自噬相关分子通路影响角质形成细胞凋亡导致创面难愈合的机制。为创面愈合中角质形成细胞对氧化损伤的应对机制提供新的研究思路。

AOPPs（晚期氧化蛋白产物）是氧化应激反应的标志性产物之一，参与多种疾病的病理生理反应。我们的前期研究首次探究了AOPPs在人角质形成细胞（HaCaT细胞）氧化应激反应中的作用，结果显示AOPPs在糖尿病等慢性疾病患者皮肤组织内的含量较正常人群明显升高，进而可能促进角质形成细胞凋亡，这一机制可能是导致慢性创面愈合延迟的原因之一。有趣的是，我们在上述研究过程中发现了HaCaT细胞自噬反应。然而，AOPPs对HaCaT细胞自噬的影响尚不清楚。本研究首次探索AOPPs诱导人角质形成细胞自噬的过程，结果提示AOPPs可通过mTOR/Beclin-1通路诱导角质形成细胞（HaCaT细胞）发生自噬反应，提示在AOPPs相关的氧化应激反应刺激下，细胞自噬可能是细胞凋亡的重要调节因子，也是调控延迟愈合创面的病理生理机制的原因之一。这部分是本课题的主要研究成果，相关文章已在投稿阶段。本课题组拟进行体内实验来验证现有的研究结果，后期我们会利用项目现有的充足条件及项目结余资金，启动动物体内实验相关研究的设计与实施，进一步优化并深入本课题组的研究。综上，项目组通过本课题的开展，发现AOPPs通过自噬相关分子通路影响角质形成细胞凋亡导致创面难愈合的机制。为创面愈合中角质形成细胞对氧化损伤的应对机制提供新的研究思路。