THE DISPOSITION AND MOVEMENT OF CHOLESTEROL IN CELLS

本研究的目的在于阐明 哺乳动物细胞内的胆固醇。 指导这项工作的大前提是 未酯化的细胞胆固醇存在于多个离散的池中， 它们不能混合。 (1)第一个目标是验证从以前的研究中得出的结论 人成纤维细胞质膜几乎包含所有的 未酯化的胆固醇。 两相水分配 为此目的，已经开发了纯化质膜的技术。 这是为了证明细胞胆固醇更丰富， 这些级分比通用细胞表面标记物5 ′-核苷酸酶。 (2)细胞内胆固醇的小池将与 大量的背景膜胆固醇通过氧化 后者选择性地在完整细胞中与胆固醇氧化酶一起。 的分数 细胞内胆固醇的溶酶体将建立 通过使用两相水溶液分析纯化的级分， 分配和平衡蔗糖梯度离心。 (3)胆固醇运动的动力学将被用来检查是否 来自胆固醇酯液滴储存的细胞内胆固醇， 摄入的脂蛋白和从头生物合成移动到质膜 通过共同的路径或通过多个非混合路径。 同样地， 细胞内胆固醇向质膜的转移 将在大鼠卵巢颗粒细胞中检查与类固醇激素产生的关系 细胞 (4)在一项平行研究中，将使用亚细胞分离技术 研究是否有胆固醇来自三个来源， (3)以上混合或保持分离。 毛地黄皂苷将被用作一种特异性的 浮力密度变化试剂来检测富含甾醇的膜。 (5)使用胆固醇氧化酶，假设细胞紧密连接 培养物中的上皮细胞单层构成了 顶侧和基底侧质膜之间的胆固醇将 考察

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.