VSV通过上调GSDME蛋白诱发细胞焦亡的机制研究

水疱性口炎病毒（VSV）具有高度的嗜上皮性，感染的上皮细胞呈现明显的凋亡现象，但机体的炎性反应仍然十分强烈。本项目前期研究发现，焦亡相关蛋白GSDME在被感染的上皮细胞中显著上调，推测细胞焦亡是VSV诱导炎性反应的重要原因，但其相关机制尚不清楚。基于此，本项目拟通过敲除和过表达Gsdme基因，明确GSDME在VSV诱导焦亡中的作用；利用RNAi阻断caspase-3/8及颗粒酶B功能，阐明GSDME的激活途径；利用免疫共沉淀及质谱技术，筛选VSV感染后与GSDME发生互作的因子，并进一步鉴定影响GSDME稳定性的因子；通过亚硫酸盐测序及染色质免疫共沉淀，解析VSV感染后Gsdme转录调控区DNA甲基化及组蛋白修饰的变化，筛选鉴定参与调控的表观修饰酶。预期结果不仅有利于揭示VSV的促炎性致病机理，而且可为VSV活病毒载体及相关产品的安全应用提供理论指导。

Vesicular stomatitis virus (VSV) is highly epidermotropic, the infected epithelial cells show obvious apoptosis, but the inflammatory response of the body is still very strong. In the previous study of this project, pyroptosis-related protein GSDME was significantly up-regulated in the infected epithelial cells. It is speculated that cell pyroptosis is an important cause of VSV-induced inflammatory response, but the related mechanism is unclear. Based on this, this project intends to clarify the role of GSDME in VSV-induced pyroptosis through knocking out and overexpressing of Gsdme gene; clarify the way of GSDME activation by RNAi functional blocking of caspase-3/8 and granzyme B; screen the factors that interact with GSDME after VSV infection by immunoprecipitation and mass spectrometry, and further identify the factors that affect the stability of GSDME; Through sulfite sequencing and chromatin immunoprecipitation, the changes of DNA methylation and histone modification in the transcriptional regulatory region of Gsdme after VSV infection were analyzed, and the enzymes involved in the regulation were screened and identified. It is expected that the results will not only help to uncover the pro-inflammatory mechanism of VSV, but also provide theoretical guidance for the safe application of VSV vector and related products.