TGF-β/PAI-1/ECM信号通路在巨噬细胞介导的弥散性放射性肺损伤中的机制研究

Radiation-induced pulmonary injury（RIPI）is a severe dose limiting toxicity reaction of radiotherapy in thoracic cancer patients. Sporadic radiation pneumonitis has recently drew quite attention because of its poor therapeutic efficacy and high mortality; however, its exact molecular mechanism is still unclear. TGF-β, as a key mediator, is strongly related to the development of pulmonary fibrosis. Most recent studies have shown that the fibrinolytic system of PAI-1 and PA was regulated by TGF-β, the disruption of whose balance would contribute to the development of pulmonary fibrosis through promoting ECM accumulation. Our previous studies showed that bystander macrophages could be activated after co-culture with irradiated pulmonary epithelial cells, with the secretion of important inflammatory factors, including TGF-β、IL-6 and TNF-α. Furthermore, the inflammation and myosization of non-targeted lung tissues could be induced by partial lung tissues irradiation of mice, where abscopal effects were mediated through the infiltration of a large number of tissue macrophages. This project focuses on urgently solving the clinical barrier, and based on the previous studies, a cell co-culture system and animal model of bilateral radiation-induced lung injury were used to investigate the intercellular signaling networks between multi-cell types that occur in RIPI, to reveal the regulation effect of key immune cell macrophages and TGF-β/PAI-1/ECM signaling pathway in promoting the formation of radioactive inflammatory microenvironment and fibrosis. The project would offer help to exploit new potential pharmacological therapeutic targets for relieving RIPI.

放射性肺损伤是影响胸部肿瘤放疗疗效和预后的重要剂量限制性毒性反应，近年来弥散性放射性肺损伤因其治疗疗效不佳且患者病死率高而逐渐受到重视。TGF-β是肺纤维化的关键调节因子，TGF-β上调可破坏纤溶酶原激活物（PA）与其抑制剂PAI-1的动态平衡，进而导致细胞外基质（ECM）大量积聚，最终发展为肺纤维化。申请者前期的研究结果表明，体外接受照射的肺上皮细胞能够激活未受照射巨噬细胞，诱导其炎性因子TGF-β、IL-6及TNF-α的释放进而介导辐射旁效应现象；体内小鼠局部肺组织受照射后能够诱导未受照肺组织炎症及纤维化反应，并伴有大量巨噬细胞的浸润参与介导辐射远端效应。本项目拟在前期研究的基础上，建立多细胞共培养体系和局部受照后双侧肺损伤小鼠模型，探究TGF-β/PAI-1/ECM信号通路在免疫巨噬细胞诱导的弥散性放射性肺损伤中的机制，为挖掘潜在的药理学治疗靶点提供研究基础。

通过构建重症放射性肺损伤小鼠模型，研究发现重症模型处理组相对于药物或辐照单因素处理，HE和Masson染色显示肺组织更严重的炎性浸润和间质肺纤维化。辐照2m后，重症模型组小鼠不仅受照射右肺组织炎症及实变，未受照射左侧肺组织也表现为明显炎症及纤维化实变。6m后小鼠肺功能检测显示模型处理组VC、FVC和FEV50等参数均较单处理组更低，表明小鼠肺通气功能受到严重损伤。RNA-seq数据分析显示重症肺损伤模型组与药物或者放疗诱导的肺损伤在基因表达水平改变上存在一定共同性，差异基因功能富集到共同的信号通路，通过体外验证显示，小鼠肺上皮细胞MLE-12和人肺上皮细胞Beas-2b细胞P21基因的表达上调明显，并参与上皮细胞对巨噬细胞U937的趋化，进一步研究发现上皮细胞CC7、CXCL9、CXCL14和CXCL16表达上调，敲低P21后CCL7的表达下调，表明上皮细胞中P21促进CCL7的分泌。本项目在博来霉素诱导的小鼠肺间质性疾病的基础上加以辐照，通过成功构建重症弥散性放射性肺损伤小鼠模型，我们观察到小鼠死亡率、炎症或纤维化程度都较单因素处理组更为严重，该模型能够较好地模拟临床伴有间质性肺病的肺癌患者接受放疗后肺部放疗后损伤加重甚至死亡的现象。我们的研究发现上皮细胞与巨噬细胞是重症肺损伤DAD及进展性肺纤维化发生发展中的关键组成，受损肺上皮细胞衰老蛋白P21升高参与免疫系统激活，通过调控CCL7的表达和分泌趋化巨噬细胞到达受损部位并向M1型极化，本研究成功构建重症肺损伤小鼠模型，有利于进一步探索肺损伤生物标志物及有效药物。