组织定居巨噬细胞对肿瘤相关巨噬细胞形成的贡献及其分子调控机制研究

Macrophages (MΦ) are divided into two essential subpopulations, namely, tissue resident macrophages (MΦ-T) and inflammatory macrophages (MΦ-I), according to their anatomical locations and functional phenotypes. It has been demonstrated that tumor-associated macrophages (TAMs) play versatile roles in the development and progression of cancers. Our previous studies have shown that Notch signaling regulates macrophage activation and the formation of TAMs. However, whether MΦ-T can be converted into MΦ-I and participate in the formation of TAMs has not been elucidated. It is also unclear whether and how Notch signaling regulates the proliferation of MΦ-T and their conversion into MΦ-I. In the current project, we intend to clarify the contribution of MΦ-T and MΦ-I to the formation of TAMs by using GFP transgenic mice and bone marrow transplantation approaches. We will also reveal the function of Notch signaling pathway in the regulation of TAM formation from different types of macrophages using a macrophage-specific knockout mouse strain of RBP-J, the critical transcription factor of Notch signaling. Furthermore, with advanced cellular and molecular biology methods, we will investigate the molecular mechanisms of Notch-mediated regulation of TAM formation, and moreover, from this point, to look into the regulatory network of MΦ-T converting into MΦ-I during TAM formation. These studies are expected to uncover the molecular mechanisms of MΦ-T differentiating into MΦ-I, and the cell sources and regulatory mechanisms of TAM formation. The results will provide new strategies and targets for the intervention of TAMs, therefore it should have important theoretical significance and potential application value.

巨噬细胞(MΦ)基于解剖位置和功能表型分为静息状态下执行吞噬功能的组织定居MΦ(MΦ-T)和炎症刺激下参与免疫应答的骨髓单核来源的炎性MΦ(MΦ-I)。肿瘤相关MΦ(TAM)在肿瘤发生发展中发挥广泛作用。我们前期证实Notch信号调控MΦ活化和TAM形成，但Notch信号是否调控MΦ-T增殖及向MΦ-I转化，其分子机制和生物学意义都有待进一步回答。本课题拟采用GFP转基因小鼠和骨髓移植实验，明确实体瘤生长中MΦ-T和MΦ-I对TAM的贡献；采用Notch信号关键转录因子RBP-J的MΦ特异性基因剔除小鼠，揭示Notch信号对不同来源的MΦ形成TAM的调控作用；采用细胞和分子生物学研究手段，阐明Notch信号调控不同来源的MΦ形成TAM的分子机制和调控网络。本研究不仅有望揭示MΦ-T向MΦ-I转化的分子机制，还可明确TAM形成的细胞来源及其调控机制，从而为TAM的靶向干预提供新的思路和靶点。

巨噬细胞(Mφ)基于解剖位置和功能表型分为静息状态下执行吞噬功能的组织定居Mφ(Mφ-T)和炎症刺激下参与免疫应答的骨髓单核来源的炎性Mφ(Mφ-I)。肿瘤相关Mφ(TAM)在肿瘤发生发展中发挥广泛作用。本项目的前期研究证实Notch信号调控Mφ活化和TAM形成，但Notch信号是否调控Mφ-T增殖及向Mφ-I转化，其分子机制和生物学意义都有待进一步回答。本课题拟采用GFP转基因小鼠和骨髓移植实验，明确实体瘤生长中Mφ-T和Mφ-I对TAM的贡献；采用Mφ特异性剔除Notch信号关键转录因子RBP-J小鼠，揭示Notch信号对不同来源的Mφ形成TAM的调控作用；采用细胞和分子生物学研究手段，阐明Notch信号调控不同来源的Mφ形成TAM的分子机制和调控网络。. 我们利用髓系细胞特异剔除/激活Notch信号的小鼠和对照小鼠，建立肝内接种肝癌、结直肠癌转移和皮下Lewis肺癌等肿瘤模型，发现巨噬细胞敲除RBP-J小鼠的皮下肺癌体积明显小于对照小鼠，但在原位肝癌模型中巨噬细胞剔除RBP-J小鼠的肝肿瘤体积显著大于对照小鼠。由于Notch信号缺失阻断骨髓来源单核细胞向肿瘤相关巨噬细胞（TAMs）分化。该结果提示：在肝脏中组织定居巨噬细胞，即Kuppfer细胞（KCs）或许参与了TAMs的形成并影响肿瘤的进展。的确，当利用巨噬细胞剔除RBP-J小鼠结合单核来源巨噬细胞阻断的CCR2剔除小鼠建立原位肝癌模型时，我们发现巨噬细胞剔除RBP-J小鼠肝癌组织中TAMs的数量显著增加。增多的TAMs来源于KCs的贡献。进一步的体内、体外机制研究显示，巨噬细胞Notch信号缺失一方面通过激活Wnt/β-catenin信号途径促进KC-TAM增殖，另一方面通过调控Wnt信号下游c-Myc的表达使其向M2型TAMs 转化。同样，巨噬细胞Notch信号缺失促进结肠癌向肝脏快速转移。TAMs中Notch和Wnt信号呈负相关性在肝癌病人组织中得到进一步验证，且Wnt信号高表达与肝癌病人预后差成正相关。对Notch信号途径调控巨噬细胞M1/M2极化的非编码RNA机制进行研究发现了一批Notch信号相关的miRNA。本项目的研究为临床靶向不同来源巨噬细胞治疗肿瘤提供了新思路和新靶点。.