基于TLR4相关的MAPK/NF-κB信号通路探讨茯苓多糖对肺癌免疫调节作用的分子机制

Nowadays, radiotherapy and chemotherapy is the primary clinical treatment of lung cancer which is the highest incidence of malignant tumor. But it is poor curative effect due to the decline of immune function. Pachyman (PHY) is often used to adjuvant therapy in the treatment of tumors as enhance immunity. And yet, the specific mechanism of PHY is unclear yet. The project will investigate the molecular mechanism of PHY from the signaling pathways. We know the expression of multiple nodes in TLR4\ MAPK\NF-κB pathway have obvious differences between lung cancer tissue and normal tissue through bioinformatics analysis. Besides this, our preliminary study found PHY can affect the expression of some genes and proteins in Toll-Like Receptor 4(TLR4) signaling pathway. As complex network formed between each signal pathway, we suppose that the mechanism of antineoplastic activity of PHY thought immunoregulation may be mediated by TLR4 and the relevant MAPK/NF-κB signal pathway. This project intends to adopt the mice model of lung cancer and cultivate of it’s Mouse Peritoneal Macrophages (MPM) in vitro. The Flow cytometry, laser confocal, quantitative PCR, Western blot, ELISA and some other experimental techniques supplemented are applied to explore the immunoregulatory anti-tumor effects of PHY and the influence on some related signal pathways on the basis of the bioinformatics analysis. Along with the channel blockers and the genetic defects tumor-bearing mice for verification from the other hand. In order to fully disclose the mechanism of immunomodulatory anti-tumor effects of PHY, and provide a credible theoretical basis for PHY in structural modification and clinical use.

肺癌是当前发病率最高的恶性肿瘤，主要以放、化疗及手术治疗为主，但疗效不佳，且损伤机体免疫功能。茯苓多糖（PHY）可提高免疫力，常用于肿瘤的辅助治疗，但具体机制不明。我们通过生物信息学分析可知TLR4及MAPK、NF-κB信号通路中多个节点在肺癌与正常组织之间的表达存在明显差异。本课题组前期研究发现PHY可影响TLR4通路相关节点基因和蛋白的表达水平。基于各通路间复杂的网络联系，我们设想PHY免疫调节抗肿瘤活性可由TLR4及其相关的MAPK/NF-κB通路介导。本项目拟采用肺癌小鼠腹腔巨噬细胞培养模型和荷瘤鼠模型，经生物信息学分析，运用流式细胞计数、激光共聚焦、Q-PCR和Western blot等技术，探究PHY免疫调节作用及其分子机制，并利用通路阻断剂和基因缺陷小鼠肺癌模型进行反向验证。拟全面揭示PHY免疫调节抗肿瘤作用机制，为PHY结构改造及临床应用提供可信的理论依据。

肺癌是目前发病率最高的恶性肿瘤之一，主要以放、化疗、手术及靶向治疗为主，但疗效不佳，且损伤机体免疫功能。茯苓多糖（PHY）可提高免疫力，常用于肿瘤的辅助治疗，但具体机制不明。我们通过生物信息学分析发现TLR4及MAPK、NF-κB信号通路中多个节点在肺癌与正常组织之间的表达存在明显差异。本课题组前期研究发现PHY可影响TLR4通路相关节点基因和蛋白的表达水平。基于各通路间复杂的网络联系，我们设想PHY免疫调节抗肿瘤活性可由TLR4及其相关的MAPK/NF-κB通路介导。基于上述基础，我们采用体外RAW264.7巨噬细胞和小鼠肺癌动物模型进行验证。本研究获得以下结果：（1）在体外建立小鼠巨噬细胞培养模型，发现PHY能显著促进巨噬细胞IL-2, IL-6, IL-17A, TNF和IFN-γ细胞因子的分泌；（2）PHY对NO、第二信使Ca2+离子的释放具有显著地促进作用；（3）在肺癌荷瘤小鼠体内实验发现，PHY表现出显著减轻瘤重和增加免疫器官指数的作用；（4）PHY在体内实验中具有促进外周血中细胞因子的分泌作用；（5）在体内外试验中均发现，PHY对TLR4信号转导途径中关键节点的基因及蛋白有显著地影响；（6）在基因缺陷小鼠体内及体外RAW264.7细胞模型的阻断剂实验中验证了PHY的免疫调节抗肿瘤的机制。由此证实，TLR4相关的MAPK/NF-κB信号可能是APS发挥免疫调节抗肿瘤作用的信号通路之一。 在本项目的资助下，已发表SCI文章5篇。在此期间培养研究生5名，使研究人员具备更好的实验技能和科研思维。本项目的研究成果，揭示了PHY免疫调节抗肿瘤作用的机制，为免疫中药机制的探讨提供了研究思路及技术平台，已达到技术目标。