靶向干预胰腺癌微环境中肿瘤浸润淋巴细胞和肿瘤细胞的Toll样受体4对胰腺癌增殖、侵袭和转移的机制研究

Tumor's proliferation, invasion and metastasis is closely related to the complex cells component and extracellular matrix in the tumor microenvironment. Tumor-infiltrating lymphocytes(TIL), the major kind cells in the microenvironment, could inhibit tumor cells' proliferation, invasion and metastasis in vitro distinctly whereas not obvious in vivo, and the mechanisms for such a big difference are still remain unknown. Our previous study have demonstrated that the Toll-like receptor 4(TLR4) widely expressed on the surface of pancreatic carcinoma cell and TIL, and down-regulation of the expression of pancreatic carcinoma cell's TLR4 in tumor cells inhibited the proliferation of the tumor cell obviously. However, how TLR4 molecules on different cell surface influence each other? What's the mechanism influencing the pancreatic carcinoma cells' proliferation, invasion and metastasis through regulating TLR4's expression on pancreatic carcinoma cells/TIL respectively or simultaneously? On the basis of preliminary study, this project is designed to establish pancreatic cancer model in TLR4 wide type and TLR4 mutants' type mouse, separate and extract primary pancreatic carcinoma cell/TIL and stimulate TLR4 alone or simultaneously, in order to observe the biological behaviors' difference and study its mechanisms. Also we inject TIL, the expression of TLR4 on which has been regulated, into the pancreatic cancer mouse model through tail vein, further to investigate the significance of TLR4 for TIL to influence pancreatic cancer's biological behaviors. Consequently, all of the above will provide new theoretical basis for the immunotherapy of pancreatic cancer.

肿瘤微环境中多种细胞及基质的相互作用与肿瘤的生长、侵润和转移密切相关，其中肿瘤侵润淋巴细胞（TIL）为一大类细胞，在体外培养扩增后能明显的杀伤肿瘤细胞但体内作用不明显，其作用的差别如此之大除了体内难以达到体外实验所需的细胞数量外还有其它机制不得而知。我们的研究发现胰腺癌细胞和TIL均能表达Toll样受体4(TLR4)，抑制癌细胞TLR4的表达能明显抑制胰腺癌细胞的增殖。但胰腺癌细胞和TIL表面TLR4的表达如何相互影响？分别或同时调控其表达如何影响胰腺癌细胞的增殖、侵袭和转移及机制如何？为此，本课题拟在前期研究基础上建立TLR4野生型和缺失型小鼠胰腺癌模型，分离胰腺癌细胞和TIL，单独或同时刺激其上的TLR4，观察对胰腺癌细胞生物学行为的影响及机制，同时将调控TLR4后的TIL尾静脉回输入胰腺癌小鼠，观察对胰腺生物学行为的影响并探讨其机制。从而为胰腺癌的免疫治疗提供理论依据。

胰腺癌之所以治疗效果差，根源在于其复杂的肿瘤微环境，其中的巨噬细胞不但没有起到免疫杀伤肿瘤细胞的作用，反而为肿瘤的生长、浸润、转移提供支持。本研究发现，正是肿瘤相关巨噬细胞（TAM）表面的Toll样受体4（TLR4）在TAM促进胰腺癌肿瘤微环境中缺氧的发生、促进胰腺癌的生长、侵袭中起到了关键作用。首先，我们通过细胞间接共培养实验发现，与缺失了TLR4的TAM共培养后，胰腺癌细胞的细胞增殖、侵袭能力的下降；其次，我们探寻TLR4的缺失对TAM内NF-kB的影响：发现缺失TLR4的TAM，其NF-kB的p65亚基的含量减少、NF-kB调控的下游因子TNF-a的mRNA相对表达量减少，释放到细胞外到TNF-a的浓度下降；再次，我们发现缺TAM缺失TLR4后，胰腺癌细胞内NF-kB的活性下降、HIF-1a的mRNA的相对表达量减少以及其蛋白生成减少，在阻断了TAM分泌的TNF-a后，也有相似的效应。据此我们推测，TAM的TLR4激活后，下游的NF-kB也被激活，引起TAM分泌TNF-a，作用于胰腺癌细胞后，使其NF-kB活性增高、HIF-1a生成增多，促进了肿瘤微环境中的缺氧，促进了胰腺癌细胞的生长和侵袭。另外，许多研究显示sB7-H3和胰腺癌的不良预后相关，越来越多的证据表明sB7-H3和NF-kB在肿瘤的进展过程中存在某种联系。研究中，我们首次发现了sB7-H3能够通过TLR4/NF-kB信号通路促进胰腺癌的侵袭和转移。sB7-H3在表面mB7-H3阳性表达的胰腺癌细胞系内高表达，外分泌的sB7-H3显著促进了胰腺癌细胞系内NF-kB的活性及其转移和侵袭性。我们进一步证实了sB7-H3上调了胰腺癌TLR4的表达，激活NF-kB后促进其下游的IL-8及VEGF的表达。相反，通过ssh RNA沉默TLR4后，上述效应消失。综上，本研究具体研究了肿瘤相关巨噬细胞与胰腺癌细胞相互作用的分子机制，并阐明了TLR4在其中所起到的作用以及其中重要的作用因子sB7-H3，并通过体内实验和体外实验验证这一发现，为胰腺癌的细胞免疫治疗提供了一重要理论基础。