THE DISPOSITION AND MOVEMENT OF CHOLESTEROL IN CELLS

细胞内胆固醇的分布和运动

• 批准号: 3339822
• 负责人: YVONNE LANGE
• 金额: $ 14万
• 依托单位: RUSH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-06-01 至 1992-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3339822
• 关键词: blood lipoprotein; cholesterol; cholesterol esters; density gradient ultracentrifugation; erythrocyte membrane; eukaryote; high density lipoproteins; human tissue; laboratory rabbit; lipid transport; liposomes; lysosomes; membrane lipids; ovary; phospholipids; radiotracer; scintillation counter; steroid hormone; thin layer chromatography; tissue /cell culture; tritium

The research is aimed at the elucidation of the disposition and movement of cholesterol within mammalian cells. The major premise guiding the work is that unesterified cellular cholesterol exists in multiple discrete pools which do not mix. (1) The first goal is to verify a conclusion derived from previous studies that the human fibroblast plasma membrane contains nearly all the unesterified cholesterol in the cell. A two phase aqueous partition technique has been developed to purify plasma membrane for this purpose. It is proposed to demonstrate that cellular cholesterol is more enriched in these fractions than the universal cell surface marker 5'-nucleotidase. (2) The small pool of intracellular cholesterol will be distinguished from the massive background of plasma membrane cholesterol by oxidizing the latter selectively in intact cells with cholesterol oxidase. The fraction of intracellular cholesterol carried in lysosomes will be established through the analysis of purified fractions using two phase aqueous partition and equilibrium sucrose gradient centrifugation. (3) The kinetics of cholesterol movement will be used to examine whether intracellular cholesterol arising from cholesteryl ester droplet stores, ingested lipoproteins and de novo biosynthesis moves to the plasma membrane through a common pathway or through multiple non-mixing routes. Similarly, the divergence of intracellular cholesterol heading to the plasma membrane versus steroid hormone production will be examined in rat ovary granulosa cells. (4) In a parallel study, subcellular fractionation techniques will be used to examine whether cholesterol arising from the three sources identified in (3) above mix or remain separate. Digitonin will be used as a specific buoyant density shift reagent to detect sterol-rich membranes. (5) Using cholesterol oxidase, the hypothesis that the tight junctions of epithelial monolayers in culture pose a barrier to the diffusion of cholesterol between the apical and basolateral plasma membranes will be examined.

该研究的目的是阐明 哺乳动物细胞内的胆固醇。 指导工作的大前提是 未酯化的细胞胆固醇存在于多个离散池中 哪些不混合。 (1) 第一个目标是验证先前研究得出的结论 人成纤维细胞质膜几乎含有所有 细胞内未酯化的胆固醇。 两相水分配 为此目的，已开发出纯化质膜的技术。 建议证明细胞胆固醇更富含 这些分数比通用细胞表面标记 5'-核苷酸酶高。 (2) 细胞内胆固醇的小池将与 通过氧化质膜胆固醇的大量背景 后者用胆固醇氧化酶选择性地作用于完整细胞。 分数 溶酶体中携带的细胞内胆固醇的含量将被确定 通过使用两相水分析纯化的级分 分配和平衡蔗糖梯度离心。 (3) 胆固醇运动动力学将用于检查是否 由胆固醇酯液滴储存产生的细胞内胆固醇， 摄入的脂蛋白和从头生物合成移动到质膜 通过共同途径或通过多个非混合途径。 相似地， 细胞内胆固醇向质膜的分歧 将在大鼠卵巢颗粒中检查类固醇激素的产生 细胞。 (4) 在平行研究中，将使用亚细胞分离技术 检查胆固醇是否来自于以下三种来源： (3)将以上混合或保持分开。 洋地黄皂苷将被用作特定 浮力密度位移试剂可检测富含甾醇的膜。 (5) 利用胆固醇氧化酶，假设紧密连接 培养物中的上皮单层对扩散构成障碍 顶端质膜和基底外侧质膜之间的胆固醇 检查了。