基于多组学整合分析的皂苷类疫苗佐剂作用机制研究

PD系从桔梗中筛选到的佐剂活性皂苷化合物，可诱导小鼠C2C12成肌细胞代谢重构引发炎性反应。本项目以代谢组学为切入点，结合转录组学、蛋白质组学和蛋白修饰组学，多组学整合筛选PD肌注小鼠股四头肌目标代谢物及其上游目标代谢酶和下游候选靶蛋白。通过体内外功能获得性、缺失性和挽救性实验，以及利用对应代谢酶条件性敲除小鼠研究目标代谢物的功能及在介导PD佐剂效应中的作用；检测添加目标代谢物、对应代谢酶的抑制剂和基因沉默对PD诱导细胞候选靶蛋白表达和修饰的影响，确定目标代谢物调控的靶蛋白；检测靶蛋白基因过表达、干扰及修饰位点突变对目标代谢物和PD诱导细胞炎性反应的影响，研究靶蛋白及其修饰位点功能，阐明作用机制。检测目标代谢酶基因过表达、干扰及修饰位点突变对PD诱导细胞目标代谢物生成与炎性反应的影响，结合定量代谢流分析研究目标代谢物生成、转运及其调控机制，为佐剂机制研究开辟新思路和新型佐剂设计提供新靶标。

Platycodin D (PD) is a saponin compound isolated from the roots of Platycodon grandiflorum, which can induce metabolic remodeling of C2C12 cells to elicit inflammatory response. The multi-omics (metabonomics, transcriptome, proteomics and modified proteomics) were performed to screen core target metabolites, upstream key metabolic enzymes and downstream candidate target proteins. The function of target metabolites and its role in mediating adjuvant effects were studied through in vivo and in vitro functional acquisition, deletion and salvage experiments, as well as conditional knockout mice using metabolic enzymes corresponding to target metabolites. The target proteins of key metabolites were identified through determining the effects of target metabolites, corresponding metabolic enzyme inhibitors and gene silencing on the expression and modification of candidate target proteins induced by PD. The function of target proteins and their modification sites was studied to elucidate its mechanisms of action through detecting the effects of target gene overexpression, interference and mutation of modification sites on inflammatory response induced by target metabolites and PD. The production and transport of target metabolites and their regulatory mechanisms were studied through determining the effects of overexpression, interference and modification site mutation of the upstream key metabolic enzymes on the production of target metabolites and inflammatory response induced by PD combining with quantitative metabolic flow analysis. The project was performed to open up new ideas for adjuvant mechanism research and provide new targets for new adjuvant design.