Optimisation and validation of 3D models of progressive human lung fibrosis

进行性人类肺纤维化 3D 模型的优化和验证

• 批准号: NC/V002384/1
• 负责人: Joseph Bell
• 金额: $ 14.57万
• 依托单位: University of Southampton
• 依托单位国家: 英国
• 项目类别: Fellowship
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=NC%2FV002384%2F1
• 关键词: Optimisation; validation; 3D; models; progressive

Idiopathic pulmonary fibrosis (IPF) is a disease affecting older adults which involves the build up of stiff scar tissue between cells that form the air sacs of the lung and the underlying blood vessels which transport oxygenated blood around the body. This scar tissue prevents the transfer of oxygen into the blood (and CO2 from the blood), and destroys the air sacs of the lung, leading to progressively impaired breathing and eventual death. IPF has no cure, and existing treatments only slow progression of the disease. In IPF, the cells responsible for producing scar tissue, fibroblasts, are found in small aggregates called fibroblastic foci which are thought to be the active areas of disease. Studies have also previously identified a molecule, transforming growth factor beta (TGFB) which drives fibroblasts to produce scar tissue.Animals, principally mice, have been extensively used to study IPF. Mice treated with a drug called bleomycin develop a build-up of scar tissue in their lungs like that seen in IPF. Importantly, however, this scar tissue build-up is reversible in mice, in contrast to human disease, where it is permanent and progressively worsens. Replacing these mice with a different model which better reflects IPF in humans is the goal of this project.One alternative to using animals to study disease mechanisms is growing human cells in flasks and subjecting them to various treatments to mimic how they behave within diseased tissue. However, cells in flasks grow in a single, 2D layer, in contrast to the 3D environment in the human body. Researchers in Southampton have developed a 3D fibroblast culture model which allows fibroblasts isolated from IPF lung tissue to grow more naturally. These 3D aggregates of fibroblasts resemble fibroblastic foci in the stiffened scar tissue they produce when treated with TGFB. However, while TGFB likely makes an important contribution to scar tissue formation, my previous research has identified that it is not solely responsible. For example, lack of oxygen in the densely packed fibroblastic foci also contributes to scar tissue formation, as does the presence of other mediators. In addition, my research has identified other cell types in fibroblastic foci, suggesting that the current model which just uses fibroblasts is too simplistic to accurately reflect the actual disease. The aim of this project is to develop the 3D fibrosis model to better reflect a fibroblastic focus found in IPF lung tissue. I will treat 3D cell cultures with chemicals which mimic the effects of lack of oxygen on the cells, and will add other molecules identified in IPF lung fluid to the 3D cultures to better represent the environment of fibroblast foci in IPF. I will compare the 3D culture model to fibroblastic foci from people with IPF using a technique called RNA sequencing. This quantifies the genetic messages that control how cells behave, allowing changes in cell behaviour to be identified based on the change in these gene signatures. We already have RNA sequencing data from fibroblastic foci, so we can use that to compare how well the gene signatures from the different 3D culture conditions match those in actual disease. We will also use a separate technology which allows fast quantification of these gene signatures to validate these data, in collaboration with the Medicines Discovery Catapult (MDC).After we have developed this model, we will seek to improve it further by adding other cells into it, reflecting the multiple cell types present in fibroblastic foci. This multicellular model will also be analysed by RNA sequencing and compared to fibroblastic foci. We will also collaborate with MDC to identify how the cells' genetic and metabolic signatures change spatially across the 3D model and fibroblast foci. If successful, this 3D cell culture model will give an alternative to animal models for the study of lung fibrosis and provide a novel testbed for evaluation of new treatments.

特发性肺纤维化(IPF)是一种影响老年人的疾病，它涉及形成肺气囊的细胞和在体内输送含氧血液的潜在血管之间形成的僵硬的疤痕组织。这种疤痕组织阻止氧气进入血液(以及血液中的二氧化碳)，并破坏肺部的气囊，导致呼吸逐渐受损，最终死亡。IPF没有治愈方法，现有的治疗方法只能减缓疾病的进展。在IPF中，负责产生疤痕组织的细胞，成纤维细胞，被发现在被称为成纤维细胞灶的小聚集体中，这被认为是疾病的活跃区域。此前的研究还发现了一种分子，转化生长因子β(TGFb)，它可以驱动成纤维细胞产生疤痕组织。动物，主要是小鼠，已被广泛用于研究IPF。用一种名为博莱霉素的药物治疗的小鼠，会像在IPF中看到的那样，在肺部形成疤痕组织。然而，重要的是，这种疤痕组织的积聚在老鼠身上是可逆的，而不是人类疾病，在人类疾病中，它是永久性的，并逐渐恶化。这个项目的目标是用一种更能反映人类IPF的不同模型来取代这些小鼠。使用动物研究疾病机制的另一种选择是在烧瓶中培养人类细胞，并对它们进行各种治疗，以模拟它们在患病组织中的行为。然而，烧瓶中的细胞在单一的2D层中生长，与人体的3D环境形成对比。南安普敦的研究人员已经开发出一种3D成纤维细胞培养模型，可以让从IPF肺组织中分离出来的成纤维细胞更自然地生长。当用TGFb治疗时，这些成纤维细胞的3D聚集体类似于硬化的瘢痕组织中的成纤维细胞灶。然而，尽管TGFb可能对瘢痕组织的形成做出了重要贡献，但我之前的研究发现，它并不是唯一的责任。例如，密集的成纤维细胞病灶中的缺氧也有助于瘢痕组织的形成，其他介质的存在也是如此。此外，我的研究还在成纤维细胞病灶中发现了其他类型的细胞，这表明目前仅使用成纤维细胞的模型过于简单，无法准确反映实际疾病。该项目的目的是开发3D纤维化模型，以更好地反映IPF肺组织中发现的成纤维细胞病灶。我将用模拟缺氧对细胞影响的化学物质处理3D细胞培养，并将IPF肺液中识别的其他分子添加到3D培养中，以更好地代表IPF中成纤维细胞病灶的环境。我将使用一种名为RNA测序的技术将3D培养模型与IPF患者的成纤维细胞病灶进行比较。这量化了控制细胞行为的遗传信息，允许根据这些基因签名的变化来识别细胞行为的变化。我们已经有了成纤维细胞病灶的RNA测序数据，所以我们可以用它来比较不同3D培养条件下的基因签名与实际疾病中的基因签名的匹配程度。我们还将使用一种单独的技术，与药物发现弹射器(MDC)合作，快速量化这些基因签名来验证这些数据。在我们开发了这个模型后，我们将寻求通过在其中添加其他细胞来进一步改进它，以反映成纤维细胞灶中存在的多种细胞类型。这个多细胞模型也将通过RNA测序进行分析，并与成纤维细胞病灶进行比较。我们还将与MDC合作，确定细胞的遗传和代谢特征如何在3D模型和成纤维细胞病灶中发生空间变化。如果成功，这种3D细胞培养模型将为肺纤维化的研究提供一种替代动物模型的选择，并为评估新的治疗方法提供一个新的试验台。