INTRACELLULAR MOVEMENT OF CHOLESTEROL IN FIBROBLASTS

大多数真核细胞的质膜富含胆固醇， 内膜含有很少的甾醇。 建议的目标 研究是阐明这种不均匀的机制， 胆固醇的分布 一种新的测量方法， 胆固醇在各种细胞内池和血浆之间转移 膜将应用于成纤维细胞的研究。 该方法基于 胆固醇氧化酶的用途，所述胆固醇氧化酶催化 在完整细胞中，质膜胆固醇转化为胆甾烯酮， 胆固醇在内部池不变。 因此血浆中的胆固醇 细胞膜可以与细胞内分离和区分 胆固醇 放射性胆固醇的分布和转移 将在胆固醇内部标记的细胞中检查样本池 从[3H]乙酸的生物合成，并在细胞表面用 外源性[14C]胆固醇。 转移到质膜的速率 通过受体介导的放射性胆固醇进入溶酶体 胆固醇酯标记低密度脂蛋白内吞作用 也将通过该技术进行测量。 平衡密度梯度 将双标记胆固醇氧化酶处理的细胞离心， 用于检查标记在各种细胞内池之间的转移。 这种技术的力量在于它提供了一种方法， 消除质膜的大量背景贡献 通过选择性地将胆固醇转化为胆甾烯酮来实现梯度。 使用这种方法，将解决以下问题：1）什么 是新合成胆固醇的运输机制 到质膜上吗 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)是否有一个共同的途径转移到 生物合成胆固醇和胆固醇的质膜， 细胞通过受体介导的低密度脂蛋白的内吞作用？ 的 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.