Lipid and Protein Effects on Mono-Layer Stability

描述（由申请人提供）：我们假设少量（1-5 wt%）的胆固醇降低了肺表面活性剂单层中饱和脂质的结晶顺序，导致剪切粘度降低，从而增强了表面活性剂流动和覆盖肺泡界面的能力。在较高浓度下，胆固醇降低了单分子层的弹性，这反过来又导致单分子层抵抗塌陷的能力下降，导致最小表面张力升高和肺功能下降。这些假设提示了替代肺表面活性剂的最佳胆固醇含量。我们将使用我们实验室独有的宏观和微观流变仪器，通过测量临床和模型肺表面活性剂的剪切粘度和弹性作为胆固醇成分的功能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射同步加速器x射线衍射相关联，以确定胆固醇如何改变肺表面活性剂脂质的分子包装，这决定了低表面张力和快速扩散和吸附所必需的单层的机械性能。我们的目标是确定快速扩散和低表面张力的生理最佳粘度和弹性，以及如何通过控制呼吸窘迫综合征合成替代肺表面活性剂的胆固醇，脂质和蛋白质组分来最佳地实现这一最佳。除了最佳组合物外，在呼吸循环期间，必须从肺泡液中将足够的表面活性剂吸附到界面上。肺表面活性剂特异性蛋白SP-A， B和C，以及磷脂酰甘油和胆固醇等脂质，被假设为促进亚相表面活性剂和界面之间的交换。然而，很少有定量证据表明存在特定的脂质和/或蛋白质交换。同样未知的是吸附发生时的表面压力，或者吸附是否在界面压缩或膨胀时发生。为了解决这一假设，我们将利用共聚焦显微镜和多种荧光染料进行光学切片，绘制出肺表面活性成分从界面到亚相的三维分布。我们预计SP-A， B和C促进吸附；然而，我们不知道特定的脂质或蛋白质是否优先吸附在界面上，以优化在高表面压力下坍塌的单层组成。将天然SP- A， B和C与肽模拟物进行比较，以评估肽的功效。公共卫生相关性：缺乏肺表面活性物质，往往是由于早产，负责新生儿呼吸窘迫综合征（NRDS）。2002年，NRDS影响了美国约24000名新生儿，目前的治疗方法使用从动物中提取的替代表面活性剂。本研究的目的是开发一种完全合成的替代表面活性剂，以降低NRDS治疗的成本，提高均匀性，减少感染因子污染的可能性，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.