INTRACELLULAR MOVEMENT OF CHOLESTEROL IN FIBROBLASTS

大多数真核细胞的质膜富含胆固醇，而 内膜中的甾醇含量非常低。建议的目标是 研究是解释这种不一致的机制。 胆固醇的分布。一种新的测量方法 胆固醇在不同细胞内池和血浆之间的转移 膜将应用于成纤维细胞的研究。该方法基于 胆固醇氧化物酶的使用，它催化氧化 质膜胆固醇在完整细胞中转化为胆固酮，同时离开 内部池中的胆固醇未受影响。因此，血浆中的胆固醇 膜可以从细胞内分离和区分出来 胆固醇。放射性胆固醇在人体内的分布和转移 将在内部标记胆固醇的细胞中对池进行检查 从[~3H]醋酸酯生物合成并在细胞表面标记 外源性[14C]胆固醇。向质膜转移的速率 受体介导的放射性胆固醇导入溶酶体内 [~3H]胆固醇酯标记低密度脂蛋白的内吞作用 也将通过这项技术进行测量。平衡密度梯度 双标记胆固醇氧化酶处理细胞的离心法将 用于检查各种胞内池之间的标记转移。 这项技术的力量在于它提供了一种手段 全程消除质膜对背景的巨大贡献 通过选择性地将其胆固醇转化为胆固酮来实现梯度。 使用这种方法，将解决以下问题：1)什么 这一机制是否参与了新合成胆固醇的运输？ 到质膜？2)这是胆固醇引入的情况 外源进入质膜不平衡 细胞内池？3)有没有共同的途径转移到 生物合成胆固醇的质膜和进入 低密度脂蛋白受体介导的细胞内吞作用？这个 胆固醇在人类心血管疾病中的特殊作用 对这些问题的充分说明。

The plasma membrane of most eukaryotic cells is rich in cholesterol whereas internal membranes have very little sterol. The objective of the proposed research is the elucidation of the mechanisms governing this non-uniform distribution of cholesterol. A novel method for the measurement of cholesterol transfer between various intracellular pools and the plasma membrane will be applied in a study of fibroblasts. The method is based on the use of the enzyme cholesterol oxidase which catalyzes the oxidation of plasma membrane cholesterol to cholestenone in intact cells, while leaving cholesterol in internal pools untouched. Thus cholesterol in the plasma membrane can be separated and distinguished from intracellular cholesterol. The distribution and transfer of radiocholesterol between pools will be examined in cells labeled internally by cholesterol biosynthesis from [3H]acetate and labeled at the cell surface with exogenous [14C]cholesterol. The rate of transfer to the plasma membrane of radiocholesterol introduced into lysosomes by the receptor-mediated endocytosis of low density lipoprotein labeled with [3H]cholesteryl ester also will be measured by this technique. Equilibrium density gradient centrifugation of double-labeled cholesterol oxidase-treated cells will be used to examine the transfer of label between various intracellular pools. The power of this technique lies in the fact that it provides the means to eliminate the massive background contribution of plasma membrane throughout the gradient by selectively converting its cholesterol to cholestenone. Using this methodology, the following questions will be addressed: 1) What is the mechanism involved in the transport of newly synthesized cholesterol to the plasma membrane? 2) It is the case that cholesterol introduced exogenously into the plasma membrane does not equilibrate with intracellular pools? 3) Is there a common pathway of transfer to the plasma membrane of biosynthetic cholesterol and cholesterol which enters the cell by receptor-mediated endocytosis of low density lipoprotein? The extraordinary role of cholesterol in human cardiovascular disease warrants a full elucidation of these issues.