基于数字基因表达谱技术筛选流感病毒疫苗佐剂茯苓多糖的靶基因并研究其功能

Influenza virus vaccine is the main measure to prevent influenza virus infection. It is considered that influenza was caused by external evil and seasonal climates while the body was deficiency of vital qi in TCM theory. It is found that the immune effects of influenza virus vaccine could be promoted by pachman which could strengthen vital qi to eliminate pathogenic factor, but the mechanism is unclear. The research object of this study is pachman. The change of gene expression that influenced by pachman which is an adjuvant of influenza virus vaccine would be detected by Digital Gene Expression Profiling (DGE). The key target genes and main singling pathways would be screen by bioinformatics, and then be checked through the experiments in vivo. Finally, the function and regulation mechanism of target genes would be researched through molecular biological technique, and then the mechanism for enhancing the effects of influenza virus vaccine anti influenza virus should be clear. This study is combined with the experiments in vivo and in vitro that contained many kinds of advanced research techniques. This study will provide important information for establishing the experiments to screening the immune adjuvant of influenza virus vaccine, and promote the application of Traditional Chinese medicine anti influenza virus.

流感病毒疫苗是防治流感病毒感染的主要措施。流感属中医疫病范畴，为机体正气亏虚，外感疫毒之邪夹时令之气趁虚而入，中伤机体所致。研究表明，具有扶正祛邪功能的茯苓多糖能够促进流感病毒疫苗的免疫效应，但是其作用机制不清楚。本项目以茯苓多糖为研究对象，首先利用数字基因表达谱技术测定茯苓多糖作为流感病毒疫苗佐剂导致巨噬细胞的基因表达变化情况，然后利用生物信息学分析技术筛选出关键靶基因和信号通路分子，并且通过小鼠体内实验验证其准确性，最后采用分子生物学技术研究靶基因及信号通路分子的功能和调控机制，藉此探讨茯苓多糖作为流感病毒疫苗佐剂的分子机制。本研究项目体内实验和体外实验相结合，综合运用多种现代研究技术，为建立筛选中药类流感病毒疫苗佐剂的实验体系提供重要依据，促进中药抗流感病毒的应用。

研究基于数字基因表达谱分析技术筛选流感病毒疫苗佐剂茯苓多糖的差异化表达基因并鉴定其主要的靶标基因和调控机制。主要研究成果包括：证实茯苓多糖含药血清主要物质成分包括D-塔格糖、葡萄糖酸、L-谷氨酰胺、L-苏氨酸等。茯苓多糖作为流感病毒疫苗佐剂的差异化表达基因共计1466个，其中上调237个，下调1229个，涉及Toll样受体信号通路、IL-17信号通路、RNA运输通路、TNF信号通路等通路。茯苓多糖可通过调节miR-21等miRNA的表达，上调TLR3、TLR8、Ifit3等基因的转录水平和IL-1β、TNF-ɑ等细胞因子的分泌。TLR3/TLR8-IRF7信号通路是茯苓多糖增强流感病毒疫苗免疫效应的重要分子通路，为建立流感病毒疫苗中医药多糖佐剂筛选体系和挖掘中医药应用价值具有重要意义。