THE DISPOSITION AND MOVEMENT OF CHOLESTEROL IN CELLS

这项研究的目的是为了阐明生物多样性的处置和运动 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 它们不能混合在一起。 (1)第一个目标是验证以前研究得出的结论 人类成纤维细胞质膜含有几乎所有的 细胞中未酯化的胆固醇。两相水相分配 为此目的，已开发了提纯质膜的技术。 有人建议证明细胞内的胆固醇在 这些组分比通用的细胞表面标记5‘-核苷酸酶。 (2)细胞内的小胆固醇池将与 质膜胆固醇氧化的大量背景 后者选择性地存在于具有胆固醇氧化酶的完整细胞中。分数 将建立溶酶体携带的细胞内胆固醇的 用双水相对精制馏分进行分析 分配和平衡蔗糖梯度离心法。 (3)胆固醇运动的动力学将被用来检验 胆固醇酯小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 通过公共路径或通过多条非混合路线。同样， 细胞内胆固醇向质膜的发散 将在大鼠卵巢颗粒中检测类固醇激素的产生 细胞。 (4)在一项平行研究中，将使用亚细胞分离技术 为了研究胆固醇是否来自 (3)以上混合或保持分开。洋地黄素将作为特效药使用 浮力密度漂移试剂检测富含类固醇的膜。 (5)使用胆固醇氧化酶，假设 培养中的单层上皮细胞构成了扩散的障碍 顶端和基底外侧质膜之间的胆固醇 检查过了。

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.