Lipid and Protein Effects on Monolayer Stability

描述(申请人提供)：肺表面活性物质中胆固醇的最佳比例仍然存在争议；胆固醇在肺表面活性物质功能中的作用仍不清楚。当将少量胆固醇添加到DPPC或临床肺表面活性物质中时，可在不改变最小表面张力的情况下将表面剪切粘度降低数量级。我们发现，胆固醇分离成一个无序的“界面相”，降低了富含DPPC的半晶体结构域之间的线张力，这反过来又显著改变了结构域的形态。这一假设是，这种界面“润滑”流动，导致单层粘度和弹性的降低，从而增强表面活性剂的流动和覆盖界面的能力。在较高的胆固醇浓度下，我们假设相间性质消除了单层必要的单层粘聚力，从而在较高的表面张力下发生坍塌。这些观察表明，替代肺表面活性物质存在一个最佳的胆固醇含量。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垂直和水平光学切片来绘制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。

DESCRIPTION (provided by applicant): The optimal fraction of cholesterol in lung surfactants remains controversial; the role of cholesterol in lung surfactant function is still unknown. Small amounts of cholesterol, when added to DPPC or to clinical lung surfactants, reduce the surface shear viscosity by orders of magnitude, without altering the minimum surface tension. We have found that cholesterol separates into a disordered "interphase" that reduces the line tension between semi-crystalline DPPC-rich domains, which, in turn, dramatically alters domain morphology. This hypothesize that this interphase "lubricates" flow, causing the reductions in monolayer viscosity and elasticity, thereby enhancing the surfactant's ability to flow and cover the interface. At higher cholesterol concentrations, we hypothesize that the interphase properties eliminate the monolayer necessary monolayer cohesion so that collapse occurs at higher surface tensions. These observations suggest an optimal cholesterol content exists 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 vertical and horizontal 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.