白藤梨多糖佐剂作用新的细胞和分子机制研究

AEPS had strong potential to increase both cellular and humoral immune responses and elicit a balanced Th1/Th2 response, and promoted the differentiation and maturation of RAW264.7 cell by up-regulating TLR receptor via the activation of NF-NF-κB signature pathway. To delineate the novel molecular and cellar signatures in the local microenvironment, we first performed cytokine-chemokine microarray analysis with the peritoneal fluid and serum of mice treated with alum or AEPS, in the presence or absence of OVA. Next, we identified the type of innate immune system cells recruited into the peritoneal cavity and in the draining nodes, determined Ag uptake and processing by innate immune system cells recruited into the peritoneal cavity and in the draining nodes , in the draining nodes by Flow cytometry. The expression of surface antigen molecule on innate immune system cells and the ability to prime T cell was also analyzed. Furthermore, Functional effect of depleting resident or recruited DCs on T cell priming and humoral immune response induced by OVA or OVA-alum was investigated. Based on the results obtained from the experiment, the Target cell for adjuvant activity of AEPS was identified. Then, to elucidate the signal transduction mechanisms and of the molecular targets of AEPS, the Mouse Inflammasomes RT2 Profiler? PCR Array was performed to profile the expression of 84 genes related to the formation of Inflammasomes as well TLR-mediated signal transduction in with the above identified target cell of mice treated with alum or AEPS, in the presence or absence of OVA. The significantly, differentially expressed genes were further confirmed by Western blotting, electrophoretic mobility shift assay (EMSA), specific inhibitor of signal transductive molecules, etc. 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.

白藤梨多糖(AEPS)系从白藤梨中分离到的具有佐剂活性的水溶性多糖。本项目针对多糖佐剂作用机制不明的现状，研究AEPS诱导免疫应答的新的细胞和分子机制。通过检测腹腔渗出液和血清中细胞因子和趋化因子含量，鉴定募集到腹腔中免疫细胞的类型、腹腔和淋巴结中摄取抗原和佐剂、加工处理抗原的细胞群，定量分析不同时间不同类型细胞抗原的摄入量和处理量，观察细胞摄取抗原和佐剂的形式、分析腹腔和淋巴结中抗原提呈细胞的表面分子表达水平及刺激T细胞的能力，候选靶细胞的去除对AEPS佐剂活性的影响，确定AEPS佐剂作用的靶细胞。以"炎性小体"PCR芯片检测靶细胞中参与炎性小体形成、NLR受体及其信号介导的相关基因表达谱的影响，筛选出显著差异表达的基因，进行生物信息学分析，构建基因调控网络，确定AEPS佐剂作用的信号转导通路及作用的靶分子。为合理设计新型、安全、有效的佐剂和高通量筛选佐剂活性天然产物奠定基础。

白藤梨多糖(AEPS)系从白藤梨中分离到的具有佐剂活性的水溶性多糖。本项目通过检测AEPS腹腔注射后局部组织细胞因子和趋化因子含量，流式细胞术鉴定募集到腹腔中免疫细胞的类型、腹腔和淋巴结中摄取抗原和加工处理抗原的细胞群，分析淋巴结中抗原提呈细胞的表面分子表达水平，确定AEPS作用的候选靶细胞。采用小鼠表达谱芯片检测了AEPS对小鼠RAW264.7巨噬细胞和AEPS腹腔注射对局部组织基因表达谱的影响，筛选出差异表达的mRNA和长链非编码RNA (lncRNA)，采用IPA (Ingenuity Pathway Analysis)软件分析差异表达mRNA的核心通路、功能、上游调节因子、调节效应以及生物学网络。通过lncRNA与mRNA关联分析预测了差异表达lncRNA的靶基因和转录因子，构建“lncRNA–转录因子–靶基因”三元组关系网络图。在此基础上，对新发现的一个正向免疫调节linRNA——lincRNA-AR1开展了系统的鉴定和功能分析研究，阐明lincRNA-AR1的序列结构信息、特性及生物学功能。本项目研究结果为今后研究免疫活性多糖分子新靶点及高通量筛选免疫活性多糖奠定良好的基础和技术支撑。

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