Pathophysiological studies in neonatal tracheal aspirate derived mesenchymal stromal cells and animal models to determine the role of Pbx1 and other candidate genes in congenital diaphragmatic hernia.

对新生儿气管抽吸物衍生的间充质基质细胞和动物模型进行病理生理学研究，以确定 Pbx1 和其他候选基因在先天性膈疝中的作用。

• 批准号: 461188606
• 负责人: Privatdozent Dr. Richard Wagner
• 金额: --
• 依托单位: Klinik und Poliklinik für Kinderchirurgie
• 依托单位国家: 德国
• 项目类别: WBP Fellowship
• 财政年份: 2021
• 资助国家: 德国
• 起止时间: 2020-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/461188606?language=en
• 关键词: Pathophysiological; studies; neonatal; tracheal; aspirate

Our goal with this proposal is to understand the role of the transcription factor Pbx1 in the pathophysiology of congenital diaphragmatic hernia (CDH) using cell-based and animal models. CDH is characterized by a diaphragmatic defect that occurs during embryonic development and is associated with pulmonary hypoplasia and persistent pulmonary hypertension. The high mortality (30-50%) of affected neonates depends largely on the degree of pulmonary hypoplasia, of which the underlying pathobiology is not sufficiently understood. We know from genome wide association studies, that transcription factor Pbx1 is a candidate gene in CDH and knockdown of Pbx1 in mice results in pulmonary hypoplasia and hypertension as seen in CDH patients. Pbx1 is expressed until the postnatal period and direct stimulation of Pbx1 downstream targets can rescue compromised lung development in affected lungs. Therefore, it is potentially relevant as a therapeutic target for postnatal interventions. Here, we aim to describe the role of Pbx1 in normal and abnormal lung development due to CDH. We will use mesenchymal stromal cells isolated from tracheal aspirates from intubated neonates with CDH and “lung healthy” controls and perform different mechanistic studies. We will leverage CRISPR/Cas9 mediated knockdown of Pbx1 in these mesenchymal stromal cells and test how this affects cell morphology and determine changes in epigenetic signature of these cells. Moreover, we will obtain proteomic profiles of the respective cells at different developmental timepoints to analyze the changes on protein level. As they emerge other candidate genes will be tested accordingly with the established work pipeline. Finally, CDH tracheal aspirate cells will be subjected to gene replacement or treatment with drugs designed to regulate or perturb pathways identified by protein interaction analyses.

我们的目标是了解转录因子Pbx 1在先天性腹股沟疝（CDH）的病理生理学中的作用，使用基于细胞和动物模型。CDH的特征在于胚胎发育期间发生的肺发育缺陷，并与肺发育不全和持续性肺动脉高压相关。受影响的新生儿的高死亡率（30-50%）在很大程度上取决于肺发育不全的程度，其潜在的病理生物学还没有充分了解。我们从全基因组关联研究中得知，转录因子Pbx 1是CDH的候选基因，小鼠中Pbx 1的敲低导致肺发育不全和高血压，如在CDH患者中所见。Pbx 1表达直到出生后，直接刺激Pbx 1下游靶点可以挽救受影响肺部受损的肺发育。因此，它作为产后干预的治疗靶点具有潜在相关性。在这里，我们的目的是描述的作用Pbx 1在正常和异常的肺发育由于CDH。我们将使用间充质基质细胞分离气管吸出物从插管新生儿CDH和“肺健康”对照，并进行不同的机制研究。我们将利用CRISPR/Cas9介导的这些间充质基质细胞中Pbx 1的敲低，并测试这如何影响细胞形态，并确定这些细胞表观遗传特征的变化。此外，我们将获得不同发育时间点的相应细胞的蛋白质组谱，以分析蛋白质水平的变化。当它们出现时，其他候选基因将通过已建立的工作管道进行相应的测试。最后，将对CDH气管吸出物细胞进行基因置换或用设计用于调节或干扰通过蛋白质相互作用分析鉴定的途径的药物处理。