肿瘤TLR介导内源性非可控性炎症机制对肝癌生长及NK细胞固有免疫的调控作用

Increasing evidences have set up a tight relationship between tumor and inflammation. Chronic nflammation, if not ready to resolve,then stalls for a prolonged periodcan develop into nonresolving inflammation, which is a major driver of diseases especially cancers. The hallmark for most, if not all, solid tumors is the present of significant immune cell infiltration and inflammation. Various immune cells, including T and B lymphocytes, macrophages, DC,NK cells and other cell types are frequently found to be concentrated in tumors relative to the surrounding tissue, yet it has become increasingly evident that immune cells and inflammatory processes that either precede or are subsequent to cancer development play a pivotal pro-tumorigenic role. It appears that such cells are actively recruited in response to tumor derived signals as a result of tumor evolution, but the mechanisms are still to be identified. We have proved that hepatoma cells secreted elevated IL-1b which acted in promoting tumor growth,invasion and inhibiting NK cell mediated cytolysis, indicating that hepatoma cells possess an ability to produce proinflammatory cytokine IL-1b and contribute to nonresolving inflammation that recruit immune cells. Here, we will use the ECM component,soluble form biglycan acts in hepatoma cells as an endogenous ligand of TLR2 and TLR4, to address the tumor TLR mediated endogenous inducers of sustained inflammation on growth and invasion capability of tumor cells and its contribution to tumor associated nonresolving inflammation. By analyzing TLR/BGN interaction, we also aim to reveal the critical signaling pathways (eg.NF-kB,MAPK,PI-3K) triggered by TLR endogenous ligand and major inflammasome platform and mediator molecules involved(NLRP3, Caspase-1 and IL-1b). As NK cells are the important innate immune cells responsible for tumor cell killing, infiltrated cell propotion, cytotoxicity, proliferation capability of NK cells will be assesed, activation associated surface and intracelllar molecules will be detected.Finally, Our data will add values to the hypothesis that tumor TLR mediated endogenous inflammation mechanisms acts as a critical events in sustaining nonresolving inflammation, and targeting tumor TLR associated signalings or other important mediators may open novel prevental and theapeutic anvenues.

持续存在的非可控性炎症是促进肿瘤发生发展的重要因素，导致非可控性炎症存在的关键机制亟需解析。我们前期研究发现，肝癌细胞高表达IL-1b能抑制NK活性促进自身生长，提示肿瘤自身具有诱导炎症的潜能，但其机理仍不清楚。本研究拟以肿瘤TLR(TLR2,TLR4)与基质降解产生的内源性配体BGN之间的相互作用为切入点，深入探讨肿瘤TLR介导的内源性机制在激发和维持非可控性炎症并调控肿瘤自身进展中的重要作用，全面解析内源性机制活化TLR所启动的关键信号机制及调控的关键分子（如炎性小体,Caspase-1,IL-1b），确立肿瘤通过TLR维系非可控性炎症对NK细胞固有免疫功能的调制作用和特征，以充分的证据客观评价肿瘤TLR内源性激活机制对非可控性炎症贡献，明确TLR介导内源性激活形成非可控性炎症的关键节点，从全新的角度解释非可控性炎症形成机制，为抗炎和抗癌治疗提供新的干预策略和分子靶标。

为了深入解析导致肿瘤微环境中非可控性炎症反应的分子机制，结合前期研究基础我们提出，肿瘤细胞表达的模式识别受体TLR2、TLR4可能与基质成分双肽链聚糖（biglycan，BGN）相互作用，激发肿瘤细胞分泌IL-1b，加速肿瘤生物学行为恶化。研究发现，慢性肝炎和肝癌患者体内IL-1b、BGN的水平显著高于正常健康人，且这两个因子的表达水平与疾病进展程度密切相关。癌旁组织中TLR2主要表达在胞浆中，而肝癌组织中TLR2主要表达于存在于细胞膜上，且水平显著升高。肿瘤组织和癌旁组织中TLR4的水平均低于TLR2，以胞浆表达为主，肝癌组织TLR4水平略高于癌旁组织，趋向于在细胞膜分布。肝癌组织中BGN水平高于癌旁组织，可能是患者体内BGN水平升高的原因之一。在HepG2细胞中，重组人BGN（rhBGN）能够以剂量和时间依赖性方式刺激细胞分泌IL-1b。rhBGN刺激时，过表达TLR4使细胞分泌IL-1b的水平略有升高，过表达TLR2或同时过表达TLR4后，细胞分泌IL-1b的水平显著升高，且同时过表达组细胞分泌的水平高于单独过表达TLR2的细胞。过表达BGN使HepG2细胞的迁移、浸润能力增强，过表达TLR2、TLR2+TLR4后，BGN使细胞的迁移、浸润能力都进一步增强，提示BGN主要结合TLR2，TLR4具有潜在协同效应。在数条经典的炎症相关信号通路（NF-kB p65、IkBa、JNK/SAPK、p38MAPK、STAT3）中， rhBGN刺激后，HepG2细胞中phospho-IkBa迅速增高，phospho-NF-kB p65增加也较为明显，其他通路没有显著变化。过表达TLR2、TLR4后，与母本细胞相比，rhBGN能够激发更高水平的phospho-IkBa、phospho-NF-kB p65，其他通路没有显著变化。尽管phospho-NF-kB-p65的水平有升高，但仍与phospho- IkBa的变化程度不相匹配，提示可能有NF-kB家族其他成员参与信号转导。过表达BGN使肝癌细胞在Balb/c祼鼠体内的成瘤时间缩短，肿瘤生长加快，体内NK细胞杀伤肿瘤的活性减弱。综上，我们的研究首次证实肿瘤细胞自身通过表达TLR2并结合BGN，有利于形成肿瘤局部的慢性炎症微环境，以肿瘤细胞源性的炎症机制为靶向进行干预将有助于提高临床肿瘤的治疗效果。