肺预转移小生境形成过程中前列腺素E2与VEGF相互作用的机制研究

Tumor cell metastasis is facilitated by ''premetastatic niches'' formed in destination organs(such as lung) by invading bone marrow-derived cells (BMDCs). However, the regulatory mechanisms of premetastatic niche remain poorly understood. Inflammatory pathways may mediate preparation of the premetastatic niches in the lungs. Some of these pathways-activation and/or the recruitment of certain inflammatory cells-might depend on vascular endothelial growth factor receptor(VEGF) and prostaglandin E2(PGE2).Preventing metastasis will likely require identification and blockade of additional or alternative inflammatory pathways that mediate the priming of the metastatic soil and the growth of micrometastases. PGE2 as an important inflammatory mediator acts as a positive-feedback regulator for VEGF secretion. So PGE2 may regulate the BMDCs recruitment and premetastatic niches. Our studies showed that in the premetastatic phase, VEGF and PGE2 have a synergistic effect on pulmonary inflammation in mouse models. In this study, tumor-bearing mice will be used to construct the premetastatic niches model. Using immunofluorescence staining, the pulmonary inflammation and BMDCs recruitment will be observed by confocal microscopy and flow cytometry. To investigate the crosstalk effects between VEGF and PGE2, COX-2-konck out mice and RNA interference will be applied, and tumor-bearing mice will be treated with different doses of anti-VEGF antibody and COX-2 inhibitors. Western blot and RT-PCR will be used to indentify the function of specific cell signal pathways and signal molecules. The study of effects VEGF and PGE2 vascular remodelling in premetastatic niche may impact strategies for preventing or controlling lung metastasis. Focus on the early cellular and molecular events in cancer dissemination and selectivity will likely lead to new approaches to detect and prevent metastasis at in earliest inception.

肿瘤转移前期，某些器官如肺脏易受到原发肿瘤分泌因子的调控，形成预转移小生境（premetastatic niche）但其发生机制尚未阐明。我们前期研究发现PGE2和VEGF参与转移前期的肺组织炎症反应，促进肿瘤转移。但PGE2与预转移小生境的关系，无文献报道。本课题通过肿瘤转移模型动物，建立肺预转移小生境模型；应用免疫荧光染色技术，共聚焦显微镜和流式细胞仪观察肺组织病理学改变及BMDCs在肺部的分布浸润；单独或联合应用VEGF抗体和COX-2 抑制剂，应用COX-2基因敲除小鼠模型和RNA干扰技术，研究PGE2与VEGF的相互调控作用；通过细胞实验和动物实验系统的研究VEGF和PGE2在预转移小生境形成中的作用。探讨相关的信号通路，从新的角度阐明预转移小生境形成的分子机制，为肿瘤转移防治提供新思路和理论依据。

研究背景：转移是恶性肿瘤的基本生物学特征,也是肿瘤患者死亡的主要原因。转移不仅取决于癌细胞的固有特性，而且与靶器官微环境密切相关。近年来研究表明，原发肿瘤分泌的细胞因子，能够调控靶器官的微环境，形成预转移小生境（premetastatic niche）,以适应癌细胞的定植和生存。本课题的研究目的是探讨PGE2诱导肺预转移小生境发生的相关信号通路，从而阐明预转移小生境血管改变的发生机制，为防治肿瘤转移提供新思路。研究方法:本研究采用细胞培养，动物实验及分子生物技术等体内外多种实验方法，结合免疫荧光染色、激光共聚焦研究内容分三部分：1.通过建立BALB/c小鼠和4T1乳腺癌细胞小鼠乳腺癌原位转移模型，初步界定转移前期为原位肿瘤种植后第10-12天。在RFP-4T1肿瘤细胞原位注射后7-12天，荧光显微镜检测到BMDC细胞，第14-17天发现代表转移瘤的红色荧光斑最早出现在第14天的小鼠肺部，表明原发肿瘤诱导肺预转移小生境形成，同时肿瘤转移前期时间范围被确定为4T1细胞原位种植后的第12天之前。2.用动物实验及激光共聚焦实验。研究发现肺预转移小生境血管通透性升高，Occludin和ZO-1在转移前期的小鼠肺部表达明显减少；Claudin-5表达变化不明显；Claudin-1和Claudin-4在小鼠肺组织中表达量较少，且变化并不明显。粘附连接蛋白β-catenin、E-cadherin和VE-cadherin的表达无明显变化。免疫荧光显示Occludin和ZO-1在小鼠肺部存在异常分布。3.探讨肺预转移小生境形成的分子机制 应用用蛋白组学和基因组学等方法，结论：①原发肿瘤分泌的VEGF 是诱导肺预转移小生境形成的主要因子。相关信号通路为VEGF 诱导PKC 磷酸化，进而引起肺血管内皮ZO-1表达下降。从而导致肺血管内皮细胞间紧密连接断裂，血管内皮通透性增高，BMDC细胞易于渗出聚集，形成预转移小生境。