PROTEIN-LIPID INTERACTIONS IN GALLSTONE PATHOGENESIS

这项研究提案的总体目标是定义 胆汁蛋白相互作用的分子机制 胆汁中的胆固醇小泡促进胆固醇结晶 成核、晶体生长和胆结石形成。胆固醇 在美国，胆结石占到了胆结石的70%。 并导致每年有超过500,000例胆囊切除手术 估计医疗保健成本超过50亿美元。胆结石 形成是多因素的，胆汁蛋白起关键作用 一水合胆固醇成核和生长过程中的作用 水晶。胆囊粘蛋白是粘液凝胶的主要成分 胆汁起到了粘合胆固醇的“粘合剂”的作用。 形成胆结石。粘液高分泌可能发生在 胆结石的形成为其提供了理想的生长环境 胆固醇晶体。非粘蛋白，如胆汁 免疫球蛋白和氨基肽酶N也促进胆固醇 过饱和模型和人体胆汁中的晶体成核。我们 假设这些描述不佳的蛋白质-脂质相互作用 对于胆固醇结晶成核和成核起关键作用 和生长优先发生在粘液凝胶层中 胆囊炎。 这项建议的具体目的是审查 胆汁蛋白在最早的成核步骤中，融合或 模型和人体胆汁中胆固醇-磷脂囊泡的裂解。 这将通过荧光分析囊泡融合或 利用生物物理方法对形态变化进行裂解和确认 光散射和电子显微镜等技术。 将这些检测与成核时间进行比较，将确定 囊泡-蛋白质的相互作用确实是最早的事件 晶体形成和定义成核动力学。其效果 将研究粘蛋白凝胶对囊泡扩散和运动的影响 通过与合成凝胶的比较来确定其独特的性能 粘蛋白对胆固醇溶解和成核的影响。比较 在粘蛋白凝胶中使用动态和 强迫罗利光散射将区分不同的影响 凝胶粘度和囊泡融合对运动的影响 最后，胆固醇晶体在粘蛋白和粘蛋白中生长。 合成凝胶将通过位相显微镜动态检测 模型，以确定凝胶对晶体生长的作用。这个 胆固醇供体，在模型和人体胆汁中，对晶体生长的影响 凝胶将被识别。这些研究将确定独特的 胆红素蛋白与胆固醇囊泡的相互作用 溶液和凝胶中，并定义它们对晶体的贡献 成核和胆结石生长可能导致新的治疗方法 用于治疗和预防这种常见病。

The overall goal of this research proposal is to define the molecular mechanisms by which biliary proteins interact with cholesterol vesicles in bile to promote cholesterol crystal nucleation, crystal growth and gallstone formation. Cholesterol gallstones account for 70% of gallbladder stones seen in the United States and result in over 500,000 cholecystectomies annually with an estimated health care cost in excess of $5 billion. Gallstone formation is multifactorial and biliary proteins play a critical role in the nucleation and growth of cholesterol monohydrate crystals. Gallbladder mucin the major component of the mucus gel in the gallbladder acts as the "glue" which binds plates of cholesterol to form gallstones. Mucus hypersecretion may occur prior to gallstone formation resulting in an ideal environment for the growth of cholesterol crystals. Non-mucin proteins, such as biliary immunoglobulins and aminopeptidase N, also promote cholesterol crystal nucleation from supersaturated model and human biles. We hypothesize that these poorly described protein-lipid interactions are critical for cholesterol crystal nucleation and that nucleation and growth preferentially occur in the mucus gel layer of the gallbladder. The specific aims of this proposal are to examine the effect of biliary proteins on the earliest step in nucleation, the fusion or lysis of cholesterol-phospholipid vesicles in model and human bile. This will be achieved by fluorescent assays of vesicle fusion or lysis and confirmation of morphological changes using biophysical techniques such as light scattering and electron microscopy. Comparison of these assays with nucleation time will determine if vesicle-protein interactions are indeed the earliest events in crystal formation and define the kinetics of nucleation. The effect of mucin gels on vesicle diffusion and motion will be studied and compared with synthetic gels to determine the unique properties of mucin on cholesterol solubility and nucleation. Comparison of vesicles to inert microspheres in mucin gels using dynamic and forced Raleigh light scattering will differentiate between effects of gel viscosity and vesicle fusion on movement through physiological gels.Finally, cholesterol crystal growth in mucin and synthetic gels will be examined by phase microscopy in a dynamic model to identify the role of gels on crystal growth. The cholesterol donors, in model and human bile, to crystal growth in gels will be identified. These studies will determine the unique interactions between bilary proteins and cholesterol vesicles in solution and in gels and define their contribution to crystal nucleation and gallstone growth perhaps leading to novel therapies for the treatment and prevention of this common disease.