Air/liquid interface cultures for alveolar type II cell differentiation

描述(申请人提供)：众所周知，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，对成人肺中表面活性物质产生的调节还不完全清楚。目前还没有已知的药理手段来增加体内内源性表面活性物质的产生来治疗肺部疾病。我们最近重新讨论了在II型细胞的原代培养中使用气/液界面培养物(无根尖液体)。我们发现加入根尖液后迅速(&lt；48小时)降低了表面活性蛋白水平，而去除所有根尖液后迅速(&lt；48小时)恢复了表面活性蛋白水平。这项R21拨款的目的是为了发现这种效应的机制。我们认为其机制可能是通过脯氨酸羟基酶2(PHD2)感受氧，而这种作用是由HIF1a介导的。我们发现，在深层培养中，核HIF1a和HIF2增加，HIF反应基因增加，这种作用由HIF稳定剂DMOG复制，并且这种作用被补充氧阻断。下游的变化可能包括抑制两个关键转录因子C/EBPA和SREBP-1。然而，我们注意到也可能涉及其他途径，我们将检查TTF1、FOXA2、mTOR、PPAR？以及AMPK靶基因在基因芯片实验分析中的作用。我们最初的重点将放在HIF1a、C/EBP1和SREBP-1c反应基因上，因为它们可能参与其中。我们已经显著改进了我们的II型细胞培养，所以这项建议中的所有研究都将是人类II型细胞。我们将同时讨论表面活性蛋白质和磷脂的合成。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)的去酰化-反应重构途径在人类细胞中的活性低于在啮齿动物细胞中的活性。这项研究的目的是确定成人II型细胞中表面活性物质产生的调节。这项研究还应该表明，持续的肺水肿会损害I型细胞的功能，并为急性肺损伤使用补充氧气提供了另一个理由。

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.