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

基本信息

批准号：
8279217
负责人：
ROBERT James MASON
金额：
$ 19.81万
依托单位：
NATIONAL JEWISH HEALTH
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-07-01 至 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8279217
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

项目摘要

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.

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开放至关重要。然而，成人肺中表面活性剂产生的调节尚不完全清楚。目前尚无已知的药理学方法可以增加体内内源性表面活性剂的产生来治疗肺部疾病。我们最近重新审视了空气/液体界面培养物（无顶端液体）在 II 型细胞原代培养中的使用。我们发现快速添加顶端液（<48小时）会降低表面活性剂蛋白水平，并且快速（<48小时）去除所有顶端液会恢复表面活性剂蛋白水平。 R21 资助的目的是发现这种效应的机制。我们认为该机制是脯氨酰羟化酶 2 (PHD2) 的氧感应，并且该作用是 HIF1a 介导的。我们发现，在深层培养物中，核 HIF1a 和 HIF2 增加，HIF 响应基因增加，HIF 稳定剂 DMOG 再现了该效应，并且该效应被补充氧气阻断。下游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 靶基因用于微阵列实验的分析。我们最初的重点将是 HIF1a、C/EBP1 和 SREBP-1c 反应基因，因为它们可能参与其中。我们显着改进了 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）基因表达的调节，二棕榈酰磷脂酰胆碱（DPPC）的脱酰基-再酰基化重塑途径在人类细胞中的活性低于在啮齿动物细胞中的活性。本研究的目的是明确成人 II 型细胞中表面活性剂产生的调节。这项研究还应该表明，持续的肺水肿会损害 I 型细胞功能，并为急性肺损伤时使用补充氧气提供另一个理由。

项目成果

期刊论文数量（2）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Stanniocalcin-1 is induced by hypoxia inducible factor in rat alveolar epithelial cells.

Stanniocalcin-1 由大鼠肺泡上皮细胞缺氧诱导因子诱导。

DOI：
发表时间：
2014-10-03
期刊：
影响因子：
3.1
作者：
Ito, Yoko;Zemans, Rachel;Correll, Kelly;Yang, Ivana V;Ahmad, Aftab;Gao, Bifeng;Mason, Robert J
通讯作者：
Mason, Robert J

Efficient and rapid isolation and purification of mouse alveolar type II epithelial cells.

高效快速分离和纯化小鼠肺泡 II 型上皮细胞。