氧化应激影响PCV2感染复制的自噬机制及其营养调控研究

我们先前的研究证实，氧化应激可促进猪圆环病毒2 型（PCV2）的感染复制，抗氧化营养素则可阻断氧化应激对PCV2 感染复制的促进作用，但对于其作用机理仍不清楚。相关研究表明，氧化应激可促进细胞自噬，而细胞自噬可影响病毒的感染复制，但对于氧化应激是否通过细胞自噬影响病毒感染复制尚未见报道。本项目拟通过透射电镜观察、蛋白免疫印迹、GFP-LC3 质粒转染、流式细胞术、荧光定量PCR、细胞免疫荧光、自噬关键基因（ATG-5、Beclin-1）的RNA 干扰、自噬信号通路（mTOR、PI3K）的分析等技术手段和方法，研究细胞自噬在氧化应激影响PCV2 感染复制中的作用及其可能机制，并通过添加抗氧化营养素改变细胞或机体的氧化应激状态后，从自噬的角度探讨PCV2 感染复制的营养调控机制。本项目的开展有助于阐明氧化应激影响PCV2感染复制的作用机理，并为抗氧化营养素在调控猪圆环病毒病中的应用提供依据。

Our previous studies have confirmed that oxidative stress plays an import role in enhancing PCV2 replication, and antioxidants can block the promotion of PCV2 replication induced by oxidative stress, but the related mechanisms are still not clear. It has been demonstrateded that oxidative stress can induce cell autophagy and cell autophagy can influence some virus replication, while there is still no report about the role of autophagy in the influence of oxidative stress on virus replication. The present project will investigate the role of autophagy in the influence of oxidative stress on virus replication and its mechanism by using confocal microscopy, Western Blot, transfection of GFP-LC3 Ⅰ plasmid, flow cytometry, real-time PCR, indirect fluorescence assay, knockdown on the autophagy related genes (ATG-5, Beclin-1) and the analysis in the pathway related to the autophagy (mTOR, PI3K). After the supplementation of antioxdants in vivo and in vitro, the nutritional regulation of PCV2 infection will also be studied from the point of view of cell autopahgy. This project will contribute to elucidate pathogenesis of porcine circovirus type 2 and provide a scientific basis for the use of antioxidants such as selenium in the nutritional regulation of PCV2 infection.