基于TLR4信号通路黄芪多糖促树突状细胞交叉递呈抗肿瘤作用及miRNA调控机制的研究

Function defect of dendritic cell (DC) which cannot activate cytototic T cell (CTL) to kill tumor cells is an important manifestation of Qi deficiency in cancer patients and it is closely related with the occurrence and development of cancer. Enhancing DC function based on the theory of “strengthening body resistance and eliminating evil” is an important starting point for tumor prevention and treatment with traditional Chinese and Western medicine. Astragalus Polysaccharide (APS) as the main compoment of Astragalus which is one of the typical tonic traditional Chinese medicine, has been widely used in anti-tumor therapy, however, the mechanism is still not clear. Our previous work revealed that Astragalus Polysaccharide (APS) could effectively activate CTL which lead to the killing of tumor cells through TLR4 pathway. Researches also show that, DC cross presentation of exogenous antigen is a key link in the activation of CTL which is regulated by TLR4 pathway. Therefore, the hypothesis of our study is: the anti-tumor effect of APS is by promoting DC cross presentation which induce CTL activation through TLR4 signaling pathway. Tumor bearing mice and OVA protein model are used in this project, key factors of TLR4 pathway are blocked and cross presentation inhibitors are used to understand the anti-tumor effect of APS; miRNA chip technology is used to clarify the epigenetic mechanism of APS on DC cross presentation based on TLR4 pathway. This study provides modern scientific support for the theory of “strengthening body resistance and eliminating evil” of traditional Chinese medicine, also it provides the theoretical basis for anti tumor polysaccharides of traditional Chinese medicine.

肿瘤患者存在树突状细胞（DC）功能缺陷，不能有效活化细胞毒T细胞（CTL）发挥杀伤肿瘤细胞的作用。传统中医学认为正虚邪实是肿瘤的基本病机，现代研究表明DC功能低下是机体正气亏虚的重要表现形式。黄芪多糖(APS)是具有扶正祛邪功能中药黄芪的主要有效成分，已广泛用于肿瘤治疗。我们前期工作初步证实APS可通过TLR4信号通路活化CTL杀伤肿瘤细胞，但作用机制尚不明确。研究表明DC对外源性抗原的交叉递呈是活化CTL的关键环节，受TLR4信号通路调控。因此，本课题假说为：APS通过TLR4通路促进DC交叉递呈，诱导CTL活化发挥抗肿瘤作用。课题以荷瘤小鼠和OVA蛋白模型为研究对象，通过阻断TLR4及其下游关键因子、采用交叉递呈抑制剂等，明确APS抗肿瘤作用机制，通过miRNA芯片技术，阐明APS通过TLR4通路促DC交叉递呈的表观遗传学机制，为中医“扶正祛邪”理论提供现代科学支持。

研究发现黄芪多糖(Astragalus polysaccharids，APS)可促进树突状细胞(Dendritic cells，DCs)成熟，发挥抗肿瘤作用，但具体机制尚不清楚。课题以DC2.4、CT26及OVA蛋白为研究对象，采用Q-PCR、ELISA、细胞流式、miRNA芯片及基因过表达等技术围绕TLR4及其下游信号通路深入研究APS对DC交叉递呈抗肿瘤及miRNA调控机制的影响。实验结果：①APS可促进DCs TLR4通路中关键基因TLR4、MYD88以及IKK2 mRNA的表达，且APS和TNF-α合用时效果最优；②APS可促进DCs表面CD80、CD86表达，IL-6、IL-12以及IL-23的分泌增加，DCs细胞中MHC-I类分子与OVA共定位增强，促进DCs交叉递呈，进而激活T细胞，并分泌细胞因子IL-2，下调IL-10的分泌，增强CTL对肿瘤的杀伤作用，且APS与TNF-α协同使用效果最好；加入TLR4通路阻断剂TAK-242或ST2825后，上述作用效果均明显下降。③与对照组相比，APS组mmu-miR-223-3p基因表达明显下调，经数据库筛选，mmu-miR-223-3p与TLR4通路密切相关。构建过表达mmu-miR-223-3p 的DCs，APS干预，结果显示，与空白组比较，APS组上清液中IL-2的含量无显著差异。说明APS可能是通过下调 mmu-miR-223-3p的表达促进DCs交叉递呈。④通过观察APS干预的DCs疫苗对荷瘤小鼠一般情况、抑瘤率、脏器指数以及血清中相关细胞因子的影响得出：与空白组相比，APS+TNF-α组荷瘤小鼠的脾脏指数最高，抑瘤率也最高，且血清中IL-2、IL-6、IL-12、TNF-α以及IFN-γ的含量显著升高；加入TLR4通路阻断剂后，与TNF-α组相比，APS+TNF-α组干预的DCs疫苗对荷瘤小鼠的抑瘤率、脾脏指数以及血清中IL-2、IL-6、IL-12、TNF-α以及IFN-γ含量的影响均无显著差异（P>0.05）。结论：APS可能是过TLR4介导的MYD88通路促进DCs成熟、活化，并通过下调DCs mmu-miR-223-3p的表达促进交叉递呈，激活T细胞，进而发挥抗肿瘤作用的，且APS和TNF-α协同使用时效果最优，为中医“扶正祛邪”理论提供现代科学支持，为补益中药的抗肿瘤研究提供新的理论依据。