Air/liquid interface cultures for alveolar type II cell differentiation

用于肺泡 II 型细胞分化的空气/液体界面培养物

• 批准号: 8191639
• 负责人: ROBERT James MASON
• 金额: $ 23.78万
• 依托单位: NATIONAL JEWISH HEALTH
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8191639
• 关键词: ABCA3 gene; ADD-1 protein; Acute Lung Injury; Acyltransferase; Address; Adult; Adult Respiratory Distress Syndrome; Air; Alveolar; Apical; Area; Breathing; CCAAT-Enhancer-Binding Proteins; Cell Culture Techniques; Cell Differentiation process; Cell Respiration; Cell physiology; Cells; Citrates; Complex; Culture Media; Cytoplasm; Development; Diffusion; Epithelial; Excision; Fatty Acids; Fatty-acid synthase; Gases; Gene Expression Regulation; Gene Proteins; Gene Targeting; Genes; Genomics; Goals; Grant; Human; Impairment; Lead; Lipids; Liquid substance; Lung; Lung diseases; Lysophosphatidylcholines; Mediating; Metabolic; Mitochondria; Nuclear; Oxygen; PDPK1 gene; Pathway interactions; Peroxisome Proliferator-Activated Receptors; Phospholipids; Pneumonia; Process; Procollagen-Proline Dioxygenase; Production; Proteins; Pulmonary Edema; Pulmonary Surfactants; Regulation; Rodent; Role; SLC2A1 gene; SRE-1 binding protein; Stearoyl-CoA Desaturase; System; Type I Epithelial Receptor Cell; Type II Epithelial Receptor Cell; Vascular Endothelial Growth Factors; alveolar type II cell; base; deacylation; density; factor C; human FRAP1 protein; hypoxia inducible factor 1; improved; in vivo; insight; lipid biosynthesis; new therapeutic target; novel; research study; surfactant; tissue culture; transcription factor

DESCRIPTION (provided by applicant): Pulmonary surfactant is known to be critical for gas exchange and for maintaining patency of small airways. However, regulation of surfactant production in the adult lung is not completely understood. There is currently no known pharmacologic means to increase endogenous surfactant production in vivo for the treatment of lung diseases. We recently revisited the use of air/liquid interface cultures (no apical fluid) in the primary culture of type II cells. We found that the addition of apical fluid rapidly (<48 h) reduces surfactant protein levels and removal of all apical fluid rapidly (<48 h) restores surfactant protein levels. The purpose of this R21 grant is to discover the mechanism for this effect. We propose that the mechanism is oxygen sensing by prolyl hydroxylase 2 (PHD2) and that the effect is HIF1a mediated. We find that in submerged cultures there is an increase in nuclear HIF1a and HIF2, an increase in HIF responsive genes, the effect is reproduced by the HIF stabilizer, DMOG, and that the effect is blocked by supplemental oxygen. The downstream alterations likely include inhibiting two key transcription factors C/EBPa and SREBP-1. However, we are mindful that other pathways are also likely involved, and we will examine TTF1, FOXA2, mTOR, PPAR? and AMPK target genes in the analyses of the microarray experiments. Our initial focus will be on HIF1a, C/EBP1, and SREBP-1c responsive genes, since they are likely to be involved. We have significantly improved our type II cell cultures so all the studies in this proposal will be human type II cells. We will address both surfactant protein and phospholipid synthesis. We believe that the deacylation-reacylation remodeling pathway for dipalmitoylphosphatidylchoine (DPPC) is less active in human cells than in rodent cells based on the regulation of gene expression of lysophosphatidylcholine acyltransferase (LPCAT1). The goal of this study is to define the regulation of surfactant production in adult human type II cells. This study should also indicate that sustained pulmonary edema will impair type I cell function and provide another reason for the use of supplemental oxygen in acute lung injury. PUBLIC HEALTH RELEVANCE: Alveolar type II cells produce pulmonary surfactant and surfactant is required for breathing. Although a lot is known about components of surfactant, we still do not know how to stimulate increased production in the adult lung. The novel culture system that we are using may allow for discovering the critical features for surfactant regulation. These factors could lead to the development of new therapeutic targets. These new therapies may prove very important to correcting the surfactant abnormalities present in acute lung injury and diseases of the small airways.

描述（由申请人提供）：已知肺表面活性物质对气体交换和维持小气道通畅至关重要。然而，成人肺中表面活性剂产生的调控尚不完全清楚。目前还没有已知的药理学方法来增加体内内源性表面活性剂的产生来治疗肺部疾病。我们最近重新研究了在II型细胞原代培养中使用气/液界面培养（无根尖液）。我们发现，快速添加根尖液（<48 h）可降低表面活性剂蛋白水平，快速去除根尖液（<48 h）可恢复表面活性剂蛋白水平。本R21基金的目的是发现这种效应的机制。我们认为这一机制是由脯氨酰羟化酶2 （PHD2）的氧感应作用引起的，其作用是由HIF1a介导的。我们发现，在潜水培养中，核HIF1a和HIF2增加，HIF反应基因增加，这种效应被HIF稳定剂DMOG复制，并且这种效应被补充氧阻断。下游的改变可能包括抑制两个关键转录因子C/EBPa和SREBP-1。然而，我们注意到其他途径也可能参与其中，我们将检查TTF1， FOXA2, mTOR， PPAR？和AMPK靶基因在微阵列实验中的分析。我们最初的重点将放在HIF1a、C/EBP1和SREBP-1c应答基因上，因为它们很可能参与其中。我们已经大大改进了II型细胞培养，因此本提案中的所有研究都将是人类II型细胞。我们将讨论表面活性剂蛋白和磷脂的合成。我们认为，基于溶血磷脂酰胆碱酰基转移酶（LPCAT1）基因表达的调控，DPPC在人细胞中的去酰基-酰化重塑通路的活性低于啮齿动物细胞。本研究的目的是确定成人II型细胞中表面活性剂产生的调节。这项研究还表明，持续的肺水肿会损害I型细胞的功能，并为急性肺损伤中使用补充氧提供了另一个理由。