基于诱导宿主细胞释放DNA的皂苷佐剂作用机制研究

TSA is a triterpenoid saponin compound isolated from the roots of Polygala tenuifolia Willd. TSA had strong potential to increase both cellular and humoral immune responses and elicit a balanced Th1/Th2 response in mice. The cytotoxic effects of TSA on the muscle cells, its effect on the expression levels of the key genes related to apoptosis, pyroptosis and necroptosis, and its induction of DNA released from the muscles in mice were first determined. We also determined the effect of TSA on serum antigen-specific antibody titers, the antigen-specific proliferation of OVA-stimulated splenocytes and the cells in the lymph nodes, the contents of cytokines in the culture supernatants of OVA-stimulated splenocytes, and the intracellular cytokine response of CD4+ and CD8+ T cell in OVA-immunied mice in the presence and absence of DNase I. Next, we investigated the levels of proinflammatory cytokines at the injection sites, identified the types of innate immune cells recruited into the quadriceps, determined Ag uptake and processing by innate immune cells recruited into the quadriceps and in the draining nodes in OVA-immunied mice in the presence and absence of DNase I by Flow cytometry. The expression of surface antigen molecule and the ability to prime T cell of dendritic cells in OVA-immunied mice in the present and absent of Dnase I was also analyzed. Then, to elucidate the signal transduction mechanisms and the molecular targets of TSA, the SurePrint G3 Mouse Gene Expression Microarrays (8×60 K) was performed to profile the expression of mRNA and lncRNA in the quadriceps of mice treated with OVA, OVA+TSA, or OVA+TSA-DNA in the presence or absence of DNase I. The significantly, differentially expressed genes were further confirmed by Quantitative real-time PCR. And, a comprehensive network analysis was conducted to mine the functional association between the experimentally defined sensitive genes. The project was performed to provide a reference for the study on new molecular targets of immunological adjuvants and the high throughput screening of novel adjuvants.

TSA是从中药远志中筛选到的皂苷化合物。本项目拟基于诱导宿主细胞释放DNA (TSA-DNA)开展TSA佐剂作用机制研究。通过分析细胞死亡情况、死亡途径以及DNA释放量，探讨TSA诱导的细胞死亡与DNA释放之间的关系。通过检测特异性抗体效价、抗原特异性刺激淋巴细胞增殖反应、细胞因子分泌能力以及T细胞细胞因子反应，评价TSA-DNA在TSA佐剂效应中的作用。通过分析炎性因子含量，免疫细胞募集、抗原摄取、加工处理、提呈能力，研究DNase I处理对TSA诱导先天性免疫应答的影响。采用小鼠表达谱芯片筛选四头肌中表达差异的mRNA和lncRNA，分析“lncRNAs–转录因子–靶基因”三元关系，筛选潜在lncRNA，以siRNA和TALENs技术验证其功能，鉴定与皂苷佐剂作用相关的信号转导通路及信号分子，揭示皂苷佐剂作用的分子机制，以期发现介导佐剂效应的新基因，为佐剂作用机制研究开辟新思路和新型佐剂设计提供新靶标。

通过分析细胞死亡情况、死亡途径以及DNA 释放量，探讨TSA诱导的细胞死亡与DNA 释放之间的关系。通过检测特异性抗体效价、脾细胞增殖反应、NK细胞活性、卵清蛋白免疫小鼠迟发性超敏反应以及肌注部位免疫细胞募集情况，评价TSA-DNA在TSA佐剂效应中的作用。采用小鼠成肌C2C12细胞为模型，检测细胞增殖、细胞死亡类型、促炎性因子基因和蛋白表达水平以及基因表达谱变化，研究桔梗皂苷D佐剂活性涉及的潜在信号传导途径。桔梗皂苷可以诱导caspase-1依赖性的细胞毒和瞬时炎症反应。原位局部阻断caspase-1可降低桔梗皂苷D对卵清蛋白的佐剂作用。机制研究发现桔梗皂苷D可显著提高C2C12细胞胞内Ca2+水平，促进JNK和p38磷酸化从而激活caspase-1。采用小鼠表达谱芯片检测二种皂苷（TSA和AJS）单独或联合不同抗原肌注诱导局部组织转录组的时效变化，以加权基因共表达网络分析，鉴定皂苷特异性基因模块，筛选皂苷特异性lncRNA，通过lncRNA-DNA结合trans机制筛选lncRNA的潜在靶基因，预测lncRNA的生物学功能，阐明皂苷佐剂作用的体内机制及信号转导特点。发现了一个新的lncRNA—lncRNA-SC，采用RACE方法和克隆确定lncRNA-SC的的全长序列，体外翻译系统分析蛋白编码能力，质核分离技术进行定位分析，采用获得性和缺失性研究方法揭示了其生物学功能。LncRNA-SC是一条位于小鼠17号染色体、全长为1480 nt、含二个内含子的lncRNA，其能调控C2C12细胞的增殖、死亡、迁移以及炎症应答基因的转录。