PI3K/Akt信号转导通路激活尘肺肺泡巨噬细胞的作用机制研究

In pneumoconiosis, the activation of alveolar macrophages (AM) is a symbolical event for pulmonary inflammation, and promotes the later development of pulmonary fibrosis. Detailed knowledge of the activation process is important not only for understanding the pathogenesis but also for developing therapeutic drugs. In earlier studies, we found that AM from patients of pneumoconiosis displayed significantly stronger activation characteristics (cytokine secretion, phagocytosis, etc.); in addition, in high-throughput screening of compound libraries, inhibition of PI3K or Akt was highly effective in inhibiting the activation phenotypes of AM, thus revealing a key role of the PI3K/Akt signal transduction pathway in the process. Based on a case-control model using whole lung lavage from stage-observation and stage-III patients of pneumoconiosis, we use specific compounds and siRNA for the targeted inhibition of pathway components and downstream effectors such as the various classes or subtypes of PI3K, Akt, and mTOR, so as to identify the association between the pathway signaling markers and the activation of AM; in both the cellular and the animal models, we evaluate the effects of the regulatory mechanism on pulmonary fibrosis; we also apply RNA-seq to analyze the transcriptome before and after AM activation. All of the above investigations together aim to elucidate the regulation and mechanism of AM activation by the PI3K/Akt signal transduction pathway in patients of pneumoconiosis. The research would also provide scientific evidence and support for the clinical translation of the discoveries.

肺泡巨噬细胞的活化是尘肺炎症反应的标志事件，促进后期肺纤维化的发展，因而深入研究该活化过程对发病机制的理解和治疗药物的研发都至关重要。在前期研究中，我们发现尘肺病源的肺泡巨噬细胞呈现明显的活化特征（增强的细胞因子分泌和细胞吞噬作用等），而在化合物库的高通量筛选中，抑制PI3K或Akt能高效抑制肺泡巨噬细胞的活化表型，揭示出PI3K/Akt信号转导通路的重要作用。基于接受全肺灌洗治疗的尘肺观察期与III期患者构建的病例对照模型，本项目通过特异性化合物和siRNA靶向抑制通路内部及下游的各类型PI3K、Akt、mTOR等成员，鉴定通路信号标记与细胞活化的关联，并在细胞和动物模型中评价该调控机制对肺纤维化的影响，及应用RNA-seq对比分析肺泡巨噬细胞活化前后的转录谱。相关研究旨在阐明PI3K/Akt信号转导通路激活尘肺肺泡巨噬细胞的作用机制，为临床转化提供科学依据。

以尘肺为代表的肺纤维化类型疾病是一类不可逆的且尚缺乏有效治疗手段或对症药物的肺部免疫应激疾病，缺乏明确的生物标记物作为病情发展的分子标志，进而限制了靶向小分子化合物筛选对抑制或缓解肺纤维化的研究前景。本项目建立了标准化的从肺纤维化患者全肺灌洗液中分离富集细胞集群的临床和实验技术方案；收集冻存了较大数量病例的病源细胞样本库；对病源细胞开展了基因组分型测序，鉴定不同批次样本的遗传背景；利用基因表达和免疫荧光成像分析病源细胞，鉴定了以巨噬细胞为主的特征，并利用脂多糖处理探索了病源细胞应激活化状态前后的变化；进行特异性抑制PI3K/Akt信号转导通路且可重复性高的RNA-seq实验，对病源细胞开展差异表达分析和功能通路富集分析；在肺纤维化小鼠模型中探索了特异性抑制PI3K/Akt对肺部病变形态和肺部组织细胞生理状况的比较。项目研究发现PI3K/Akt信号转导通路在肺纤维化病变中具有重要的调控作用，并揭示与该通路作用相关联的潜在分子机制和基因通路靶点。另一方面，阐明了PI3K/Akt信号转导通路在病源肺泡巨噬细胞中的调控机制及其关联的重要功能基因和胞内通路，发现了潜在的新型生物标记物，相关成果对肺纤维化靶向抑制的探索具有应用价值和临床意义。