Nemeth Julia, Schundner Annika, Quast Karsten, Winkelmann Veronika E, Frick Manfred
Institute of General Physiology, Ulm University, Ulm, Germany.
Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany.
Front Physiol. 2020 Oct 9;11:567675. doi: 10.3389/fphys.2020.567675. eCollection 2020.
Idiopathic pulmonary fibrosis (IPF) is a fatal disease of the lower respiratory tract with restricted therapeutic options. Repetitive injury of the bronchoalveolar epithelium leads to activation of pulmonary fibroblasts, differentiation into myofibroblasts and excessive extracellular matrix (ECM) deposition resulting in aberrant wound repair. However, detailed molecular and cellular mechanisms underlying initiation and progression of fibrotic changes are still elusive. Here, we report the generation of a representative fibroblast reporter cell line (10-4A ) to study pathophysiological mechanisms of IPF in high throughput or high resolution live cell assays. To this end, we immortalized primary fibroblasts isolated from the distal lung of Sprague-Dawley rats. Molecular and transcriptomic characterization identified clone 10-4A as a matrix fibroblast subpopulation. Mechanical or chemical stimulation induced a reversible fibrotic state comparable to effects observed in primary isolated fibroblasts. Finally, we generated a reporter cell line (10-4A ) to express nuclear blue fluorescent protein (BFP) under the promotor of the myofibroblast marker alpha smooth muscle actin () using CRISPR/Cas9 technology. We evaluated the suitability of 10-4A as reporter tool in plate reader assays. In summary, the 10-4A cell line provides a novel tool to study fibrotic processes to gain new insights into the cellular and molecular processes involved in fibrosis formation and propagation.
特发性肺纤维化（IPF）是一种致命的下呼吸道疾病，治疗选择有限。支气管肺泡上皮的反复损伤导致肺成纤维细胞活化，分化为肌成纤维细胞，并过度沉积细胞外基质（ECM），从而导致异常的伤口修复。然而，纤维化变化起始和进展的详细分子和细胞机制仍然不清楚。在这里，我们报告了一种代表性的成纤维细胞报告细胞系（10-4A ）的产生，用于在高通量或高分辨率活细胞测定中研究IPF的病理生理机制。为此，我们将从Sprague-Dawley大鼠远端肺分离的原代成纤维细胞永生化。分子和转录组学特征鉴定克隆10-4A为基质成纤维细胞亚群。机械或化学刺激诱导出一种可逆的纤维化状态，类似于在原代分离的成纤维细胞中观察到的效应。最后，我们使用CRISPR/Cas9技术生成了一个报告细胞系（10-4A ），以在肌成纤维细胞标志物α平滑肌肌动蛋白（ ）的启动子下表达核蓝色荧光蛋白（BFP）。我们在酶标仪测定中评估了10-4A 作为报告工具的适用性。总之，10-4A 细胞系提供了一种研究纤维化过程的新工具，以深入了解纤维化形成和传播所涉及的细胞和分子过程。
