多糖调控角质形成细胞自噬的分子机制及其抗紫外线损伤效应的研究

Several polysaccharides have been revealed for the induction effect on keratinocyte autophagy in a MTOR-independent way by our preliminary work. There are extensive associations between autophagy and maker changes caused by ultraviolet-induced damage. A variety of autophagy-related proteins and signaling pathways are involved in autophagy regulation. In this project, keratinocytes will be treated with several polysaccharides in vitro, and we aim to explore in further the mechanism of polysaccharides modulating keratinocyte autophagy through the experiment of western blot, co-immunoprecipitation and confocal laser scanning microscope tracing protein location, etc. In further, keratinocytes will be challenged by ultraviolet B (UVB) and then treated with these polysaccharides to investigate on the changes of series of events including cellular activities, migration and apoptosis, etc. This research is aimed to reveal the molecular mechanism of modulating keratinocyte autophagy and the effect of anti-ultraviolet damage of polysaccharides.

前期工作鉴定出多种多糖类物质对角质形成细胞自噬的非MTOR依赖性诱导效应。自噬与紫外线损伤的标志分子事件具有广泛的关联。自噬调控涉及多种自噬相关蛋白和信号通路的参与。本研究拟对体外培养的角质形成细胞进行几种多糖类物质孵育，采用蛋白印迹、免疫共沉淀和激光共聚焦显微镜分析蛋白定位变化等方法进一步鉴定调控自噬的机制。进而，对UVB损伤的角质形成细胞给予多糖孵育，分析细胞活力、迁移和凋亡等分子事件的反馈性变化。通过本研究可以初步分析多糖调控角质形成细胞自噬的分子机制，初步明确这些物质的抗紫外线损伤效应。

自噬是一种对于维持细胞内环境稳定极为重要的生理现象，已经被证明与许多人类疾病的发生和发展具有密切关联，以自噬调控为靶点可对潜在药物进行评估。我们的前期工作已经证明海藻糖可以通过非MTOR依赖途径诱导角质形成细胞自噬，在本项目中，我们系统研究了其它多糖（棉子糖）对UVB照射的角质形成细胞自噬和死亡的影响。建立了细胞自噬抑制和紫外线损伤模型，同时证明了自噬机制与角质形成细胞迁移的关联性，发现了与自噬和迁移调控相关的细胞内分子标志ZO-1和claudin-1；评价了多种多糖对UVB照射诱导的角质形成细胞IL-8分泌的影响。最重要的是，我们发现了参与海藻糖调控角质形成细胞自噬的两个关键基因ATG9A和TIMP3,并初步证明了这两个基因和皮肤日光性角化和鳞癌发生发展的关联性。本项目明确了海藻糖自噬调控相关的药物作用靶点，阐明了自噬机制在多糖等天然物质发挥药理活性中所起到的作用；同时丰富了非经典自噬领域机制研究的理论知识，拓展了以自噬为靶点的抗紫外线损伤研究。