应用质谱成像技术原位表征肺癌组织区域的巨噬细胞免疫特性

The mutual transformation between anti- and pro-inflammation of macrophages in different pathological environments implies that the interactions between macrophages and their surrounding medium molecules may play important roles. This study plans to establish an novel technological platform to characterize immunological characteristics of macrophages in lung cancerous tissues based on our own established mass spectrometry imaging. To in-situ investigate metabolites of macrophages and their surrounding medium molecules in lung cancerous tissues; To establish the metabolic regulation networks between metabolome of macrophages and their surrounding medium molecules; To probe the relationships between metabolites of macrophages, their surrounding medium molecules, and pathological environments. Measuring the metabolome-related enzymes and phenotypes, distribution and immunological functions of macrophages in different environments using immunohistochemical assays and phenotypes and immunological functions of induced in vitro macrophages and macrophages from tissues using flow cytometry and ELISA assays is to obtain the relationships between important metabolic molecules of macrophages and immunological functions under different environmental conditions and to recognize the plasticity and polarizability of macrophages in different pathological environments in macrophage metabolome point of view. Obtaining the important molecules and molecular profiling which involve in the anti-and pro-inflammatory processes of macrophages would allow us to deeply understand the link between macrophage heterogeneity and occurrence of cancers and to deeply understand pro-tumor mechanisms of macrophages.

病理微环境中巨噬细胞的抗炎与促炎相互转化，预示其与周围介质分子的相互作用密切相关。本研究将利用质谱成像技术，建立表征组织器官区域巨噬细胞免疫特性的新技术;原位检测肺癌组织区域巨噬细胞代谢物及其周围介质分子,从代谢组角度探究不同病理微环境区域巨噬细胞代谢组与其周围介质分子的分子调控网络,研究巨噬细胞代谢物及其周围介质分子与病理微环境特性之间的关系；利用免疫组化方法检测微环境中代谢合成酶和巨噬细胞表型、分布和免疫功能；利用流式细胞仪检测体外诱导巨噬细胞和来自病理微环境的巨噬细胞表型和免疫功能，获得不同环境下巨噬细胞代谢关键分子与其免疫特性的关系，从巨噬细胞代谢组的角度认识病理微环境中巨噬细胞的多可塑性和极化性，获得巨噬细胞在促炎和抗炎转化过程中的关键分子或分子谱，为深入解析巨噬细胞异质性与肺癌发生之间关系提供直接证据，为认识巨噬细胞促瘤机制提供新的理论依据。

巨噬细胞的抗炎与促炎功能的相互转化，预示其膜表型与其周围介质分子的相互作用密切相关。本研究将分子成像技术与高性能的质谱技术相结合，将组织微萃取与电超声离子化技术结合，通过优化前期研究结果研制了一种基于液体萃取电超声离子化技术的新型常压离子源，与高分辨傅里叶变换离子回旋共振质谱结合实现了对生物组织中分子的原位高质量分辨率和高空间分辨率的快速质谱成像。利用该技术平台可以获得质量精度优于1ppm和空间分辨率优于15微米的代谢分子的高准确度分子质谱成像，结合基质辅助激光解吸电离技术。研究发现：1）PE-Cer(d36:1)、PE(P-16:0/18:1)、PE(16:1/18:0)、PE(36:2) 、PE(18:1/18:0)和FAHFA(46:8)等代谢分子在不同类型巨噬细胞之间有着显著差异; 2)经典活化型巨噬细胞诱导分子对巨噬细胞的刺激强于不同肺癌微环境产生的诱导分子的刺激，表明经典活化型巨噬细胞和在不同肺癌微环境下活化的巨噬细胞可能有不同的免疫机制；3）不同肺癌微环境中M0型巨噬细胞大量释放肿瘤坏死因子α，但对M1型巨噬细胞没有显著的变化；4）肿瘤微环境中的M0型、M1型和M2型巨噬细胞膜表面的唾液酸化聚糖存在显著差异，其中M1型巨噬细胞表面唾液酸化聚糖最丰富，M0型巨噬细胞膜表面唾液酸化聚糖含量最低；5）不同类型肺癌细胞具有特异的膜脂表型。本研究揭示不同肺癌微环境中的巨噬细胞其膜脂表型不同，其免疫功能与其膜脂表型密切相关。