β-葡聚糖在树突状细胞抗乳腺癌免疫应答中的机制研究

In our previous studies, we found that β-glucan was able to induce anti-tumor immune response and inhibit breast cancer growth. Dendritic cells (DCs), the professional antigen-presenting cell in vivo, take important roles in eliciting tumor-specific cytotoxic lymphocyte (CTL). We found that β-glucan could induce DCs mature, upregulate surface markers such as CD40, CD86, MHC-II and secrete IL-12, TNF-α. The β-glucan stimulated DCs promoted T cell to Th1 differentiation and induce tumor specific CTL. These effects were dependent on dectin-1 pathway. Therefore we propose to dissect the mechanism of anti-breast cancer immune response by β-glucan. Three aims are proposed. Aim 1. Determine the mechanisms of signal transduction by β-glucan in DCs.We will use mouse bone marrow dendritic cells to determine which signaling pathways may be responsible for β-glucan mediated cytokine release and DC mature. In addition, we will examine T cell differentiation in the presence of β-glucan treatment both in vitro and in vivo. Aim 2. Determine the mechanisms of β-glucan induce anti-tumor adaptive immune responses. Aim 3. Determine the mechanisms by β-glucan modulate tumor miroenvironment. We will test the hypothesis that β-glucan modulates the tumor microenvironment toward Th1 response via regulating DCs，MDSC, Treg, tumor infiltrating macrophages.Mouse/human breast cancer cell line EO771/MCF-7 will be used as tumor model systems to dissect those mechanisms.

我们前期的研究发现β-葡聚糖（WGP）可以诱导抗肿瘤免疫应答，抑制乳腺癌荷瘤小鼠肿瘤的生长，WGP可以诱导树突状细胞（DCs）成熟，上调DCs细胞表面CD40、CD86、MHC-Ⅱ等分子的表达，并大量分泌IL-12，TNF-α等细胞因子，驱使CD4+T细胞向Th1分化，诱导肿瘤特异性细胞毒性T淋巴细胞（CTL）增殖，并且呈Dectin-1依赖性。DCs是目前所知的机体内功能最强的专职抗原递呈细胞，在肿瘤特异性免疫应答的激发和功能介导中起着重要的作用。本课题研究WGP在DCs中的信号传导通路，从分子机理上阐明发生这些生物学效应的机制；通过构建体内乳腺癌模型，比较分别用WGP诱导和未用WGP诱导的DCs抑制肿瘤生长的差异，并分析肿瘤组织和免疫器官中免疫细胞和免疫分子的不同，进一步研究WGP对肿瘤浸润相关免疫细胞的作用，阐明β-葡聚糖在DCs介导的抗乳腺癌免疫应答中的机制。

本研究以GM-CSF和IL-4体外诱导培养小鼠骨髓来源的树突状细胞。应用流式细胞术检测BMDCs表面分子的表达；qRT-PCR检测BMDC中CC趋化因子受体 mRNA的表达；Transwell和体内迁移实验检测BMDCs的趋化反应性；WB测定BMDCs内MAPKs信号通路中蛋白的磷酸化水平。结果：与对照组相比，β-葡聚糖能上调BMDCs表面分子MHC-Ⅱ类分子、CD80、CD86、CD40的表达，同时促进BMDCs分泌IL-6、TNF-α、IL-12p40，CCR7 mRNA水平增加，增强BMDCs对CCL19/CCL21的趋化反应性，并且显著增加从皮下注射部位迁移至引流淋巴结中BMDCs数目；Western Blot结果显示β-葡聚糖刺激BMDCs可引起ERK1/2、p38 MAPK的迅速磷酸化；ERK1/2抑制剂和p38抑制剂均可明显抑制β-葡聚糖诱导的BMDCs体外迁移能力增强和CCR7mRNA表达增加，但仅有ERK1/2抑制剂U0126能显著抑制β-葡聚糖引起的BMDCs表面分子CD80、CD86表达上调和细胞因子IL-6 、IL-12p40、TNF-α分泌增加，并且仅有ERK1/2抑制剂可以逆转β-葡聚糖诱导的BMDCs吞噬能力。进一步的研究发现，β-葡聚糖还能显著逆转TEDCs的免疫抑制功能，诱导CD4+T细胞和CD8+T细胞分别向Th1和CTLs分化，而抑制Treg细胞的增殖，并且可以诱导TEDCs向淋巴结迁移；体内研究发现，β-葡聚糖激发后的TEDCs显著抑制肿瘤的生长，明显增加肿瘤微环境中免疫反应细胞的浸润，减少免疫抑制细胞的数量，对其机制的初步研究发现，β-葡聚糖能显著并特异性的诱导TEDCs细胞中lnc-AL的上调表达，而lnc-AL KD能抑制β-葡聚糖对TEDCs的逆转效应。但是lnc-AL如何调控TEDCs免疫抑制功能的转变还有待阐明，本课题结合前期工作基础，研究lnc-AL在β-葡聚糖诱导TEDCs功能逆转中的作用及分子机制。结论：β-葡聚糖不仅能促进BMDCs分化成熟，还能增强其体内外迁移能力；β-葡聚糖能明显激活BMDCs内ERK1/2信号通路和p38 MAPK信号通路；β-葡聚糖通过ERK1/2和p38 MAPK信号通路调控BMDCs迁移功能和CCR7表达，但是只有ERK1/2通路参与调节BMDCs的功能成熟。