MAWBP通过调控树突状细胞在炎症性肠病炎症-肿瘤发生发展中的作用及机制研究

Inflammatory bowel disease (IBD) is a kind of nonresolving, non-specificity chronic inflammation, a precancerouse lesion, the mucosal immune regulated by dendritic cells takes critical part in the development of IBD. We have performed proteomic analysis and genomics analysis,which indicate that Serine/threonine kinase receptor-binding protein MAWBP was significantly down-regulated in UC intestinal biopsy compared with normal control both in the protein level and mRNA level (Fig. 3), we also found that MAWBP was negatively correlated with the inflammatory degree of UC (Fig. 2). Further more, we did the realtime PCR on the PBMCs from IBD patients and healthy control, MAWBP and MAWD were significantly down-regulated in UC PBMCs compared with healthy control (Fig. 4). Then we did the pre-experiment and found that overexpressed MAWBP in inflammatory-cancer cells model can regulate dentritic cells and antigen presenting cells (Fig. 1), inhibit a large group of cytokines and oncogenes in vitro. Therefore, we deduce that MAWBP could inhibit inflammation and cancer via regulating dendritic cells. In this project, we will verify the role of MAWBP through MAWBP-/-mice, AOM/Il10-/-infammatory-cancer animal model and patients’ samples in vivo. We will detect the role of MAWBP in maturation of dendritic cell, pattern recognition receptors, T lymphocyte proliferation, Th cell differentiation, inflammation index, cancer and pathway with flow cytometry, electron microscopy, MACS MicroBeads and qPCR et al, to investigate the negative role of MAWBP in the process of inflammation-cancer in IBD through regulating dendritic cells.

炎症性肠病(IBD)是一种非可控性慢性炎症，易恶变为结肠癌，其中树突状细胞介导的粘膜免疫发挥了重要作用。前期研究我们通过蛋白组学和基因组学方法，在蛋白和转录水平均发现丝氨酸/苏氨酸受体激酶结合蛋白MAWBP在UC患者肠粘膜中显著低表达，且与UC炎症程度呈负相关，预实验发现在体外炎症-肿瘤细胞模型中过表达MAWBP可抑制炎症因子和癌基因，调控树突状细胞及抗原递呈细胞的相关基因。因此我们推断MAWBP可能通过调控树突状细胞来发挥抑制炎症和肿瘤的作用，本项目拟在体内MAWBP-/-小鼠模型、AOM/Il10-/-炎症-肿瘤动物模型和临床样本中进一步证实MAWBP的功能和机制，通过流式细胞术、电镜技术、磁珠分选和qPCR等检测MAWBP对树突状细胞成熟、模式识别受体、T淋巴细胞增殖、Th细胞分化、炎症肿瘤指标及信号通路等的影响，探讨MAWBP通过介导树突状细胞对IBD炎症-肿瘤进程的负向调控作用。

炎症性肠病(IBD)是一种非可控性慢性炎症，易恶变为结肠癌，其中在炎症性肠病及恶变中具重要作用，其效应功能与其成熟的状态密切相关。前期研究我们通过蛋白组学和基因组学方法，在蛋白和转录水平均发现丝氨酸/苏氨酸受体激酶结合蛋白MAWBP在UC患者肠粘膜中显著低表达，且与UC炎症程度呈负相关，预实验发现在体外炎症-肿瘤细胞模型中过表达MAWBP可通过erk1/2通路抑制炎症因子和癌基因，调控树突状细胞及抗原递呈细胞的相关基因。因此我们推断MAWBP可能通过erk1/2通路，调控树突状细胞成熟分化并进一步发挥抑制炎症和肿瘤的作用，探讨MAWBP成为UC 癌变关键节点的可行性。首先我们成功构建了MAWBP-/-基因敲除小鼠，并进一步建立基因敲除小鼠实验性结肠炎模型及炎症相关癌变小鼠模型。通过体内及体外实验我们发现MAWBP在肠道炎症中起保护作用，一定程度上缓解了炎症及炎症相关癌变的进展。进一步机制研究我们发现MAWBP通过抑制erk1/2信号通路的激活通过调节Th17细胞及树突状细胞从而调控UC进程。此外，我们成功提取骨髓来源树突状细胞（BMDCs），其可经诱导进一步分化为对应于体内炎症状态下产生的炎性DC，具代表性且数量较多，可应用于体外实验。我们采用流式细胞仪检测技术观察MAWBP对DC抗原摄取能力的影响，Western bolt及qPCR检测MAWBP对BMDC成熟分化过程中相关信号通路的调控，qPCR及ELISA法检测其对树突状细胞分泌细胞因子的干预作用。我们发现敲除MAWBP可经erk1/2通路下调BMDC抗原摄取功能及细胞因子分泌，但与野生型相比并不显著，这可能与BMDC中存在异质性细胞有关。进一步我们成功提取了骨髓来源DC限制性祖细胞CDP，可特异性分化为cDC细胞及pDC细胞，可获取高纯度树突状细胞用以下一步研究，并意外发现MAWBP可能对DC谱系定向分化存在作用。本研究初步阐明了MAWBP通过erk1/2信号通路介导了树突状细胞成熟分化的分子机制，为MAWBP调控树突状细胞成熟分化并进一步发挥抑制溃疡性结肠炎及其恶变进程提供了更充分的科学依据。本研究将为非可控性炎症UC 癌变的发病机制探索提供理论和实践基础。