胚胎肺祖细胞移植对矽肺纤维化的疗效及作用机制研究

Pneumoconiosis is a systemic disease characterized by diffuse pulmonary fibrosis. China is the country with the most serious pneumoconiosis hazard in the world. The number of pneumoconiosis patients now ranks first in the world. Silicosis is the most common type of pneumoconiosis. The incidence of silicosis is high, and there is a lack of effective treatment drugs. Exploring and developing effective treatment measures for pulmonary fibrosis is of great social significance in the field of occupational disease prevention and treatment. With the further development of stem cell research, lung progenitor cells play a role in lung regeneration under the conditions of injury and diseases, which opens up a new way for the development of lung tissue regeneration therapy for pulmonary fibrosis. This project will focus on Sox9+ embryonic lung progenitor cells transplantation in vivo to treat silicosis fibrosis in mice, clarify the effect of Sox9+ mouse embryonic lung progenitor cells based on alveolar structure reconstruction mechanism in the treatment of silicosis fibrosis, clarify the differentiation direction and cell fate of Sox9 + mouse embryonic lung progenitor cells through lineage tracking and single cell sequencing, and to analyze the therapeutic mechanism of pulmonary fibrosis after cell transplantation combined with proteomic detection. This project aims to provide theoretical and experimental basis for the future treatment of silicosis by Sox9+ mouse embryonic lung progenitor cells.

尘肺病是以肺组织弥漫性纤维化为主的全身性疾病。我国是世界上尘肺病危害最为严重的国家，现患尘肺病人数均居世界之首，矽肺是尘肺病中最常见的类型。矽肺发病率高，缺乏有效的治疗药物，探讨和开发有效的肺纤维化治疗措施，在职业病防治领域具有重大的社会意义。随着干细胞研究的深入发展，胚胎肺祖细胞在损伤的肺组织中发挥的肺部再生修复作用，为肺组织再生疗法的发展开辟了新的途径。本项目将围绕Sox9+胚胎肺祖细胞体内移植治疗小鼠矽肺纤维化展开，阐明基于肺泡结构重建机制的Sox9+小鼠胚胎肺祖细胞治疗矽肺纤维化的效果；通过谱系追踪和单细胞测序方法，阐明Sox9+小鼠胚胎肺祖细胞移植后的体内分化方向和细胞命运，结合蛋白质组学检测，分析细胞移植后对肺纤维化的治疗机制。以期为将来釆用Sox9+小鼠胚胎肺祖细胞治疗矽肺提供理论基础和实验依据。

矽肺是在指在职业活动中长期吸入二氧化硅粉尘并在肺内潴留而引起的以肺组织弥漫性纤维化为主的全身性疾病。矽肺纤维化的病程较长，发病机制尚未完全明确，其病理过程以细胞的损伤和异常修复为特点，因此组织再生修复是逆转其病理状态的最佳选择。胚胎肺祖细胞的外源性干预，有望成为参与损伤肺组织重建的有效手段，Sox9基因可能是肺损伤后的肺组织再生修复的关键基因，因此，本研究旨在对Sox9+胚胎肺祖细胞移植治疗矽肺纤维化的可能性级初步的生物学机制进行研究。本研究通过2D、改良后的3D培养模式下的分化潜能变化观察，明确了2D培养导致Sox9表达的丧失和细胞分化发生，改良后的3D培养可较好地维持Sox9阳性表达。3D培养扩增后的Sox9+胚胎肺祖细胞通过非暴露式气管滴注回输至矽肺模型小鼠体内后，肺组织纤维化程度和炎症程度均有明显降低。对细胞回输后的肺组织进行检测发现，细胞回输2天后可检测到较多Sox9+胚胎肺祖细胞存活，14天后数量急剧下降，28天后基本消失。结合AQP5和SP-C蛋白表达检测发现，回输14天时肺组织存在Sox9+ AQP5+上皮细胞，说明3D培养的Sox9+胚胎肺祖细胞移植后能够在矽肺模型小鼠肺组织中定植并向肺泡上皮细胞分化，发挥再生修复功能。获取各组小鼠的肺泡灌洗液，提取总蛋白后进行DIA蛋白质组学检测，筛选表达差异较高的标记物进行western blot验证，同时对肺组织进行qPCR检测，结果表明，3D培养的Sox9+胚胎肺祖细胞移植后能够降低肺纤维化关键标记物的表达，其机制与TGF-β/SMAD信号通路和Wnt/β-catenin信号通路相关。

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