CELLS PROCESSING HIGH DENSITY LIPOPROTEINS

处理高密度脂蛋白的细胞

基本信息

批准号：
2415539
负责人：
EVE P REAVEN
金额：
$ 15.13万
依托单位：
PALO ALTO VETERANS INSTIT FOR RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
1985
资助国家：
美国
起止时间：
1985-04-01 至 2000-04-30
项目状态：
已结题

项目摘要

Evidence indicates that steroidogenic cells are capable of interiorizing lipoprotein-delivered cholesterol by a non-endocytic or 'selective' pathway. In this pathway cells preferentially take in lipoprotein-supplied cholesteryl esters (CE), relative to the uptake of lipoprotein apoproteins. The selective pathway has been shown to occur in a variety of human and animal cells in culture, but it is especially important in the ovary or adrenal where it delivers CE in bulk to cells which require cholesterol as a precursor for steroid hormone production. Thus, in our zeal to examine ways to reduce circulating cholesterol levels in individuals it is important to remember that some cells require large amounts of exogenous-provided cholesterol for their specific functions. Despite extensive use of the 'selective' pathway by cells, we are just now beginning to understand how the CE of this pathway is internalized and transported intracellularly. Recent acquisition of two major technical advances in the field have provided the means of obtaining important basic information on these questions. First, we have been able to trace the uptake and intracellular transport of a fluorescently labeled CE(BODIPY(R)-CE prepared as a component of reconstituted apo E free high density lipoprotein, hHDL3) in both intact and semi-intact rat ovarian granulosa cells in culture. The fluorescently tagged ligand is hydrolyzable by acid compartments (e.g. lysosomes) of the cell (but not by neutral components of the 'selective' pathway) and as such, has provided us with a powerful tool by which we can specifically trace early stages of this pathway. The present application outlines a variety of steps using this probe by which we can morphologically and biochemically determine the role of membranes, specific cell organelles, cytosolic factors, cytoskeletal components and energy to 'selective' pathway function, as well as a means of identifying sequential steps in the pathway, signaling events, etc. Of special interest also is the possibility of using genetically altered animals to isolate the 'selective' pathway (from other cholesterol uptake pathways) of steroidogenic cells. Knockout mice (missing the B/E receptor) or double knockout mice (missing both the B/E receptor and lipoproteins containing apoE) are available and are currently being tested by us to establish their 'selective' pathway characteristics. These special mice with their single cholesterol pathway can confirm earlier observations regarding the 'selective' pathway, but more importantly can identify later intracellular events in the selective pathway using a fluorescent CE marker (NBD-CE) which is hydrolyzable under neutral cell conditions, and releases NBD-labeled free cholesterol which can be subsequently visualized and traced.

证据表明类固醇细胞能够内部化通过非遗传或“选择性”降低脂蛋白的胆固醇路径。在此途径中，细胞优先服用脂蛋白供应相对于脂蛋白的摄取，胆固醇酯（CE）载蛋白。选择性途径已显示出在多种人类和动物细胞在培养中，但在卵巢或肾上腺大量将CE提供给需要的细胞胆固醇作为类固醇激素产生的先驱。因此，在我们的热情检查降低循环胆固醇水平的方法个人重要的是要记住，有些细胞需要大的其特定功能的外源性提供的胆固醇量。尽管通过细胞广泛使用“选择性”途径，但我们刚刚开始了解该途径的CE的内在化和细胞内运输。最近收购了两个主要的技术该领域的进步提供了获得重要基本的手段有关这些问题的信息。首先，我们能够追踪荧光标记的吸收和细胞内转运 CE（Bodipy（r） - 作为重组Apo e Free High的组成部分准备的密度脂蛋白，HHDL3）在完整和半直大的大鼠卵巢中培养中的颗粒细胞。荧光标记的配体是可通过细胞的酸室（例如溶酶体）水解（但不能 “选择性”途径的中性组件），因此提供了我们拥有强大的工具，我们可以专门追踪这条路。本应用程序概述了使用的各种步骤我们可以在形态和生化上确定的探测器膜，特定细胞细胞器，胞质因子的作用，细胞骨架成分和“选择性”途径功能的能量，作为以及识别途径中的顺序步骤的手段，信号传导事件，等等特别感兴趣的也是使用遗传改变的可能性动物分离“选择性”途径（来自其他胆固醇吸收类固醇细胞的途径）。敲除小鼠（缺少B/E受体）或双基因敲除小鼠（缺少B/E受体和脂蛋白可以使用apoe），目前正在我们测试建立他们的“选择性”途径特征。这些特殊的老鼠凭借其单一胆固醇途径可以确认早期的观察结果关于“选择性”途径，但更重要的是以后可以识别使用荧光CE的选择性途径中的细胞内事件标记（NBD-CE），可在中性细胞条件下水解，并且释放NBD标记的自由胆固醇，随后可以看到并追踪。