Lipid and Protein Effects on Mono-Layer Stability

描述(由申请人提供)：我们假设小部分(1-5wt%)胆固醇减少了肺表面活性物质单层中饱和脂肪的结晶有序性，导致剪切粘度降低，从而增强了表面活性物质流动和覆盖肺泡界面的能力。在较高浓度下，胆固醇降低单层弹性，进而导致单层抵抗坍塌的能力降低，导致更高的最低表面张力和肺功能下降。这些假设表明，替代肺表面活性物质的最佳胆固醇含量。我们将使用我们实验室独有的宏观和微观流变学仪器，通过测量作为胆固醇组成函数的临床和模型肺表面活性物质的剪切粘度和弹性来确定最佳胆固醇含量。这些机械性能将与等温线、荧光和原子力显微镜以及掠入射同步X射线衍射相关联，以确定胆固醇如何改变肺表面活性物质脂类的分子堆积，这决定了低表面张力和快速再铺展和吸附所需的单分子膜的机械性能。我们的目标是确定快速铺展和低表面张力的生理最佳粘度和弹性，以及如何通过控制用于治疗呼吸窘迫综合征的合成替代肺表面活性物质的胆固醇、脂肪和蛋白质组分来最好地实现这一最佳。除了最佳的组成，在呼吸周期中，必须从肺泡液中将足够的表面活性剂吸附到界面上。肺表面活性物质特有的蛋白SP-A、B和C，以及磷脂酰甘油和胆固醇等脂类，被假设为加强亚相和界面上表面活性物质之间的交换。然而，几乎没有关于特定的脂肪和/或蛋白质交换的定量证据。同样未知的是在什么情况下发生吸附的表面压力，或者在界面压缩或扩张过程中是否发生吸附。为了解决这一假设，我们将使用共聚焦显微镜和多种荧光染料的光学切片绘制出从界面到亚相的肺表面活性物质组分的三维分布。我们期望SP-A、B和C促进吸附；然而，我们不知道特定的脂类或蛋白质是否优先吸附到界面以优化在高表面压力下坍塌的单层组成。天然的SP-A、B和C将与多肽模拟物进行比较，以评估多肽的疗效。公共卫生相关性：肺表面活性物质缺乏，通常是由于早产，是新生儿呼吸窘迫综合征(NRDS)的原因。2002年，美国估计有24000名新生儿受到NRDS的影响，目前的治疗方法使用的是从动物中提取的替代表面活性物质。这项研究的目标是开发一种完全人工合成的替代表面活性物质，以降低NRDS的治疗成本，改善一致性，降低感染因素污染的可能性，并提高与胎粪吸入或急性肺损伤相关的RDS的治疗效果。

DESCRIPTION (provided by applicant): We hypothesize that small fractions (1-5 wt%) of cholesterol reduce the crystalline ordering of saturated lipids in lung surfactant monolayers, leading to a reduction in the shear viscosity, which enhances the surfactant's ability to flow and cover the alveolar interface. At higher concentrations, cholesterol reduces the monolayer elasticity, which in turn, leads to a decrease in the ability of the monolayer to resist collapse, leading to higher minimum surface tensions and a decrease in lung function. These hypotheses suggest an ptimal cholesterol content for a replacement lung surfactant. We will determine this optimal cholesterol content by measuring the shear viscosity and elasticity of clinical and model lung surfactants as a function of cholesterol composition using macro- and micro- rheology instruments unique to our laboratory. These mechanical properties will be correlated with isotherms, fluorescence and atomic force microscopy, and grazing incidence synchrotron X-ray diffraction to determine how cholesterol alters the molecular packing of lung surfactant lipids, which determines the mechanical properties of monolayers necessary for low surface tensions and rapid respreading and adsorption. Our goal is to determine the physiologically optimal viscosity and elasticity for rapid spreading and low surface tension and how best to achieve this optimum by controlling the cholesterol, lipid and protein fractions of a synthetic replacement lung surfactant for respiratory distress syndrome. In addition to an optimal composition, sufficient surfactant must be adsorbed to the interface from the alveolar fluid during the respiratory cycle. The lung surfactant specific proteins SP-A, B and C, along with lipids such as phosphatidylglycerol and cholesterol, are hypothesized to enhance exchange between surfactant in the subphase and the interface. However, little quantitative evidence for specific lipid and/or protein exchange exists. Also unknown is the surface pressures at what adsorption occurs, or if adsorption occurs during compression or expansion of the interface. To address this hypothesis, we will map out the three-dimensional distribution of lung surfactant components from the interface to the subphase using optical sectioning with a confocal microscope and multiple fluorescent dyes. We expect that SP-A, B and C promote adsorption; however, we do not know if specific lipids or proteins are adsorbed preferentially to the interface to optimize the monolayer composition that collapses at high surface pressures. Native SP- A, B and C will be compared to peptide mimics to evaluate the efficacy of the peptides. PUBLIC HEALTH RELEVANCE: A lack of lung surfactant, often due to premature delivery, is responsible for neonatal respiratory distress syndrome (NRDS). In 2002, NRDS affected an estimated 24,000 newborns in the US, current treatments utilize replacement surfactants derived from animals. The goal of this research is to develop an entirely synthetic replacement surfactant that should reduce costs of NRDS treatment, improve uniformity, decrease the likelihood of contamination with infectious agents, and improve the efficacy of treatment of RDS associated with meconium aspiration or acute lung injury.