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）：已知肺表面活性剂对于气体交换和维持小气道的通畅至关重要。但是，尚不完全了解成年肺表面活性剂的调节。目前尚无已知的药理学方法来增加体内内源性表面活性剂的产生，以治疗肺部疾病。我们最近重新审视了在II型细胞的主要培养物中使用空气/液体界面培养物（无顶液）。我们发现，顶端液体迅速（<48 h）的添加可降低表面活性剂蛋白水平，并迅速降低所有根尖液（<48 h）可恢复表面活性剂蛋白水平。 R21赠款的目的是发现这种效果的机制。我们提出该机理是通过羟基羟化酶2（PHD2）进行氧气传感，并且该作用是HIF1A介导的。我们发现，在淹没的培养物中，核HIF1A和HIF2（HIF响应性基因的增加）有所增加，其效果由HIF稳定剂DMOG再现，并且该作用被补充氧气阻断。下游变化可能包括抑制两个关键的转录因子C/EBPA和SREBP-1。但是，我们谨记其他途径也可能涉及，我们将检查TTF1，FOXA2，MTOR，PPAR？在微阵列实验的分析中，AMPK靶基因。我们最初的重点将放在HIF1A，C/EBP1和SREBP-1C响应基因上，因为它们可能会参与其中。我们已经显着改善了II型细胞培养物，因此该提案中的所有研究都将是人类II型细胞。我们将解决表面活性剂蛋白和磷脂合成。我们认为，基于基于溶血磷脂酰胆碱促胆碱酰基转移酶的基因表达，基于啮齿类动物细胞的二酰磷酸磷脂酰磷脂（DPPC）的脱酰化 - 丙烯酸重塑途径在人类细胞中的活性低于啮齿动物细胞（LPCAT1）。这项研究的目的是定义成人人类II型细胞中表面活性剂产生的调节。这项研究还应表明持续的肺水肿会损害I型细胞功能，并为在急性肺损伤中使用补充氧气提供了另一个原因。 公共卫生相关性：肺泡II型细胞产生肺表面活性剂，并且需要表面活性剂才能呼吸。尽管对表面活性剂的成分知之甚少，但我们仍然不知道如何刺激成人肺的产生增加。我们正在使用的新型培养系统可以发现表面活性剂调节的关键特征。这些因素可能导致新的治疗靶标的发展。这些新疗法对于纠正急性肺损伤和小气道疾病中存在的表面活性剂异常非常重要。