TLR4-NFκB在TAMs介导的动物乳腺肿瘤生长中的作用研究

As a major component of the lymphoreticular infiltration of a variety of solid tumors, tumor associated macrophages (TAMs) play important roles in the growth, progression, and metastasis of animal mammary tumor. Therefore, TAMs are being considered a logical target for treatment of mammary tumor. We have previously suggested that the number of TAMs infiltration in the tumor is correlated with the depth of invasion and micro-vessel density. Moreover, there was a positive correlation between TAMs count and clinical outcome of patients..In the present project, using cell co-culture model and mammary tumor bearing TLR4- mice model, we want to study the correlations between macrophages of M1 or M2 phenotype and mammary tumor growth and progression. Moreover, this project will aim to detect the activation of TLR4-NFκB singaling pathway and the expression of several proinflammatory cytokines including VEGF, TGFβ1, IL-6 and MMP-9. Using immunohistochemistry methods to exam whether the density of TAMs is related to mammary tumor angiogenesis and prognosis in mice bearing with mammary tumor. Macrophages have functional plasticity and can change their functional profiles repeatedly in response to environmental changes. The two extremes of polarized macrophages are named M1 and M2 macrophages. To test the transform mechanism of M1 and M2 phenotype and the correlation between TLR4-NFκB singaling pathway and this process, this project will also use M1-polarized or M2-polarized mouse macrophages to co-culture with IL-6 and IL-6 antibody. In this project, we will test the activation of TLR4-NFκB pathway and the expression of several pro-inflammatory cytokines including VEGF, TGFβ1, IL-6 and MMP-9 using Real-time PCR and Western blot methods. In addition, to clarify the correlation between TLR4-NFκB pathway and TAMs phenotype transformation, we will also analyze micro-vessel density, tumor volume and tumor metastasis..This study will clarify the effects and mechanism of different phenotype of tumor associated macrophages on mammary tumor growth and progession,and will enrich the basic theory of inflammatory tumor micro-environment promote mammary tumor progression and metastasis. This project will also provides the theoretical basis and experimental proof in the treatment of mammary tumor with different phenotype of tumor associated macrophages as therapeutic targets, and will meaningful for the study of mammary tumor immunity and tumor therapeutics.

肿瘤相关巨噬细胞（TAMs）作为肿瘤间质的主要炎症细胞，参与了乳腺肿瘤的生长和转移，因而有望成为乳腺肿瘤免疫治疗的重要靶点。我们在前期研究中已证明TAMs浸润与乳腺肿瘤恶性生长呈正相关。在此基础上，本项目拟通过构建体外细胞共培养模型和TLR4基因缺失小鼠荷瘤模型，观察肿瘤细胞凋亡、体外侵袭力、炎性细胞因子（VEGF、TGFβ1、IL-6、MMP-9）表达，检测肿瘤组织微血管密度、TLR4-NFκB蛋白及下游炎性因子的表达，分析M1、M2型巨噬细胞与乳腺肿瘤恶性生长特性的相关性，探讨TLR4-NFκB信号通路在M1、M2型巨噬细胞调节乳腺肿瘤生长及M1/M1型巨噬细胞活化亚型转化中的作用及机制。本项目预期揭示不同活化表型的TAMs对乳腺肿瘤的生长调控作用及机制，将进一步丰富癌旁炎性微环境促进乳腺肿瘤生长的基础理论。本项目的研究成果将为以TAMs为靶点的乳腺肿瘤免疫治疗提供实践依据和新的思路。

肿瘤相关巨噬细胞（TAMs）作为肿瘤间质的主要炎症细胞，参与了乳腺肿瘤的生长和转移，因而有望成为乳腺肿瘤免疫治疗的重要靶点。本项目通过构建体外细胞共培养模型和TLR4 基因缺失小鼠荷瘤模型，观察了肿瘤细胞凋亡、体外侵袭力、炎性细胞因子（VEGF、TGFβ1、IL-6、MMP-9）表达，检测肿瘤组织微血管密度、TLR4-NFκB 蛋白及下游炎性因子的表达，分析M1、M2 型巨噬细胞与乳腺肿瘤恶性生长特性的相关性，探讨了TLR4-NFκB 信号通路在M1、M2 型巨噬细胞调节乳腺肿瘤生长及M1/M1 型巨噬细胞活化亚型转化中的作用及机制。在研究中，我们发现在乳腺肿瘤细胞及组织生长过程中，肿瘤组织的微血管密度显著比空白对照组要高，差异显著；TLR4蛋白及其下游信号通路NFκB 蛋白的核转录显著增强。而使用基因敲除小鼠TLR4信号通路的阻断能显著降低肿瘤组织的微血管密度，并对M1/M1 型巨噬细胞活化亚型转化具有显著的调控作用。在项目开展过程中，我们还意外发现了多靶点作用于TLR4-NFκB通路的活性药物，如EGCG、MSC、人参皂苷Re、Rb等多种活性单体能显著调节多种乳腺肿瘤细胞凋亡、显著抑制乳腺肿瘤组织中的血管形成及调控M1/M2型巨噬细胞的表达，对肿瘤的生长具有显著抑制作用。此外，我们还发现一个特别的现象：使用具有免疫调节作用的常规治疗性药物，如丙泊酚、右美托咪啶、乙酰丙嗪、阿托品等药物进行合理的配伍后静脉或肌肉注射荷瘤鼠或荷瘤犬时，能显著抑制气管插管时机体的免疫反应及疼痛反射，并能调节手术除瘤过程中动物体的肿瘤细胞免疫清除能力。在试验开展过程中，我们还发现人参皂苷Re等活性单体不仅能够提高机体对乳腺肿瘤的清除能力，同时还具备较强的免疫佐剂作用，能提高多种疫苗包括肿瘤疫苗的免疫效果，并能调节CD31及CD80的表达，使机体接种免疫后的抗体水平显著升高，并显著延长免疫的效果。本项目揭示不同活化表型的TAMs 对乳腺肿瘤的生长调控作用及机制，进一步丰富癌旁炎性微环境促进乳腺肿瘤生长的基础理论。本项目的研究成果为以TAMs为靶点的乳腺肿瘤免疫治疗提供实践依据和新的思路。