II型肺上皮细胞衰老在放射性肺纤维化中的作用及其靶向清除治疗机制研究

Radiotherapy is an essential tool for the management of thoracic malignancies. Radiation-induced pulmonary fibrosis（RIPF） is a common disease and has a poor prognosis owing to the progressive breakdown of gas exchange regions in the lung. The etiology of radiation-induced pulmonary fibrosis (RIPF) is incompletely understood, and effective interventions are lacking. Type II pneumocytes (airway epithelial cell type II，AECII) produce surfactant and repopulate type I and II pneumocytes after insult and have been identified as stem/progenitor cell in alveolar epithelium. It has been reported that radiation induced AECII senescence may play an important role in development of RIPF. Our preliminary data showed that Bcl2 inhibitor ABT can selectively kill various senescent cells in vitro and effectively depleted senescent cell in murine bone marrow and lung after mice exposed to total body iiradiation; ABT can increase the survival and mitigated lung fibrosis after mice exposed to 17Gy thoracic X ray radiation. The role of AECII senescence and the senescence-associated secretory phenotype(SASP) will be investigated in murine lung fibrosis model induce by thoracic X ray exposure, adopting techniques such as pathological assay, fluorescence activated cell sorter(FACS), gene array and cytokine antibody array. The effects of clearance of senescent AECII and attenuation of lung fibrosis by ABT will be evaluated, the mechanism of action of ABT will be determined. Our results will provide proof that ABT will be developed as novel radiation mitigator to reduce some of radiation induced late effects. It also be beneficial to the strategies removing senescent cell to delay age-related diseases.

放射性肺损伤常见于放疗患者及辐射事故暴露人员，放射性肺纤维化（RIPF）出现在晚期，临床预后很差，缺乏有效的治疗手段。II型肺上皮细胞（AECII）是肺泡区干细胞，具有维持肺泡组织结构和功能稳定的作用。有资料显示照射可以引起AECII早期凋亡和后续的细胞衰老性损伤；预实验发现筛选出的衰老细胞清除剂Bcl2抑制剂（ABT）可以减少全身照射引起的小鼠肺组织衰老细胞；延长胸部局部照射小鼠生存期，改善肺组织纤维化。在此基础上，本项研究将通过动物和细胞试验，利用病理技术、流式细胞术、基因和抗体芯片技术，阐明衰老AECII增多及其分泌因子在RIPF发生发展中作用；观察ABT对衰老AECII的清除及减缓RIPF进展的药效学作用；探讨ABT清除衰老AECII的分子机制。本项研究提出清除放射引起的衰老组织干细胞、改善微环境将为临床治疗RILF提供新的策略；也为其他衰老相关慢性炎性疾病药物研发提供新的思路。

放射性肺纤维化是胸部肿瘤放射治疗和全身照射引起的严重损伤，目前缺乏有效治疗手段。前期研究发现细胞衰老在骨髓辐射远期损伤中起重要作用，清除衰老细胞能够显著改善造血干细胞功能。本项研究取得以下进展：1）右侧胸部局部照射引起小鼠肺纤维化及衰老性改变：小鼠17Gy右侧胸部X线照射建立了放射性性纤维化模型。研究发现受照后16周到23周模型稳定；受照后第8周开始出现细胞衰老性改变及衰老相关细胞表型（SASP）升高；验证了细胞衰老在放射性纤维化发生发展中的作用。2）ABT-263减少受照肺组织衰老细胞，减轻受照肺组织纤维化病变： 小鼠受照后16周小鼠给予2个周期ABT-263治疗，可以明显减轻受照小鼠肺纤维化改变，改变炎性因子表达水平，为衰老细胞清除应用于放射性纤维化提供了实验依据。3）ABT-263清除受照小鼠衰老II型肺泡细胞： 小鼠II型肺泡上皮细胞体外照射后出现衰老，ABT-263选择性地杀伤衰老细胞；流式分选结果发现受照肺组织II型细胞数量持续下降，放射性肺纤维化候选枢纽基因AFAP1和Nuf2减少；博来霉素诱导A549细胞衰老模型，显示AFAP1减少与SASP升高有关。4）右侧胸部局部照射影响小鼠骨髓造血功能：发现受照小鼠出现髓系细胞比例升高、淋巴细胞比例下降的细胞分化偏移现象；骨髓细胞及造血干细胞数量增加但是增殖功能下降；外周血炎性因子RANTES等水平升高，发生造血系统衰老性改变。应用ABT-263有一定保护作用。5）衰老巨噬细胞在放射性肺纤维化形成中发挥作用：发现体外培养及分选受照小鼠巨噬细胞，照射均能诱导巨噬细胞衰老和SASP分泌增加；巨噬细胞中衰老相关枢纽基因Bank1显著升高，Afap1和Nuf2显著降低；提示巨噬细胞SASP分泌增加与放射性肺纤维化的发生发展有关。本研究确认了清除衰老细胞对放射性肺纤维化的治疗作用及其机制，为临床应用提供实验依据和理论基础。