Weigle Sabine, Martin Eva, Voegtle Andrea, Wahl Benjamin, Schuler Michael
Boehringer Ingelheim Pharma GmbH & Co. KG, Department of Drug Discovery Sciences, 88397 Biberach an der Riss, Germany.
J Biol Methods. 2019 Jun 3;6(2):e115. doi: 10.14440/jbm.2019.285. eCollection 2019.
Ongoing tissue repair and formation and deposition of collagen-rich extracellular matrix in tissues and organs finally lead to fibrotic lesions and destruction of normal tissue/organ architecture and function. In the lung, scarring is observed in asthma, chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis to various degrees. At the cellular level immune cells, fibroblasts and epithelial cells are all involved in fibrotic processes. Mechanistically, fibroblast to myofibroblast transformation and epithelial to mesenchymal transition are major drivers of fibrosis. Amongst others, both processes are controlled by transforming growth factor beta-1 (TGFβ-1), a growth factor upregulated in idiopathic pulmonary fibrosis lungs. Phenotypic assays with primary human cells and complex disease-relevant readouts become increasingly important in modern drug discovery processes. We describe high-content screening based phenotypic assays with primary normal human lung fibroblasts and primary human airway epithelial cells. For both cell types, TGFβ-1 stimulation is used to induce fibrotic phenotypes , with alpha smooth muscle actin and collagen-I as readouts for FMT and E-cadherin as a readout for EMT. For each assay, a detailed image analysis protocols is described. Treatment of both cell types with TGFβ-1 and a transforming growth factor beta receptor inhibitor verifies the suitability of the assays for pharmacological interventions. In addition, the assays are compatible for siRNA and Cas9-ribonucleoprotein transfections, and thus are useful for genetic target identification/validation by modulating gene expression.
组织持续修复以及富含胶原蛋白的细胞外基质在组织和器官中的形成与沉积最终会导致纤维化病变，并破坏正常组织/器官的结构和功能。在肺部，哮喘、慢性阻塞性肺疾病和特发性肺纤维化中均会不同程度地出现瘢痕形成。在细胞水平上，免疫细胞、成纤维细胞和上皮细胞均参与纤维化过程。从机制上讲，成纤维细胞向肌成纤维细胞的转化以及上皮向间充质的转化是纤维化的主要驱动因素。其中，这两个过程均受转化生长因子β-1（TGFβ-1）调控，该生长因子在特发性肺纤维化肺中上调。在现代药物发现过程中，使用原代人类细胞进行的表型分析以及与复杂疾病相关的读数变得越来越重要。我们描述了基于高内涵筛选的原代正常人肺成纤维细胞和原代人气道上皮细胞的表型分析。对于这两种细胞类型，均使用TGFβ-1刺激来诱导纤维化表型，以α平滑肌肌动蛋白和I型胶原蛋白作为成纤维细胞向肌成纤维细胞转化的读数，以E-钙黏蛋白作为上皮向间充质转化的读数。对于每项分析，都描述了详细的图像分析方案。用TGFβ-1和转化生长因子β受体抑制剂处理这两种细胞类型，验证了这些分析方法用于药物干预的适用性。此外，这些分析方法适用于siRNA和Cas9-核糖核蛋白转染，因此可用于通过调节基因表达来鉴定/验证基因靶点。
