MOLECULAR GENETICS OF THE MACROPHAGE ACETYL LDL RECEPTOR

我们的长期目标是了解细胞的分子细节 导致动脉粥样硬化疾病的过程， 理解，以帮助制定合理的方法进行诊断， 治疗和预防动脉粥样硬化。 动脉粥样硬化斑块 含有巨噬细胞，作为清道夫细胞， 血浆脂蛋白胆固醇。 充满胆固醇的巨噬细胞 是钙泡沫细胞 本研究的目的是建立 体细胞系统的生物化学和遗传分析， 巨噬细胞特异性脂蛋白代谢和一个基因的克隆， 巨噬细胞特异性脂蛋白受体 我们以前开发了一个 将脂蛋白受体的功能基因转移到细胞中的方法 缺乏受体的细胞。 该方法结合了DNA介导的基因转移 随后用选择性培养基处理细胞。 的 选择性培养基杀死不表达脂蛋白的细胞 受体。 例如，我们使用这种方法来引入函数 人LDL受体进入LDL受体缺陷的中国仓鼠卵巢（CHO） 细胞突变体 这项建议的重点是乙酰低密度脂蛋白受体， 巨噬细胞特异性脂蛋白受体，介导 修饰的lDL和培养物中泡沫细胞的形成。 CHO细胞不 正常表达巨噬细胞特异性乙酰LDL受体。 在 初步实验，我们使用DNA转移和脂蛋白受体 选择以分离携带乙酰LDL受体的CHO细胞。 的目的 所提出的研究是详细表征脂蛋白， 所选细胞的胆固醇代谢，并比较其性质 与真正的巨噬细胞相比。 选定的单元格可能是 非常适合巨噬细胞特异性的体细胞遗传分析 脂蛋白内吞作用和泡沫细胞形成。 此外，人类 乙酰LDL受体基因将从二级DNA转染子中克隆 源自这些细胞。 人类特异性（DNA家族）重复DNA 作为探针用于检测乙酰LDL受体基因组克隆， 噬菌体文库 这些基因组克隆将依次用于分离cDNA 它将被测序以确定乙酰基的一级结构 LDL受体。 开发用于分离表达该蛋白的CHO细胞的方法， 乙酰LDL受体 开发用于分离CHO细胞的方法， 表达乙酰低密度脂蛋白受体应该被证明是有用的， 其他组织特异性受体介导的表征 内吞作用

Our long term objectives are to understand in molecular detail cellular processes which lead to atherosclerotic disease and to use this understanding to help develop rational approaches for the diagnosis, treatment and prevention of atherosclerosis. Atherosclerotic plaques contain macrophages which serve as scavenger cells that accumulate cholesterol from plasma lipoproteins. The cholesterol-laden macrophages are calfoam cells. The goal of the proposed research is the establishment of a somatic cell system for the biochemical and genetic analysis of macrophage-specific lipoprotein metabolism and cloning of a gene for a macrophage-specific lipoprotein receptor. We have previously developed a method to transfer functional genes for lipoprotein receptors into cells that lack the receptors. The method combines DNA-mediated gene transfer with subsequent treatment of the cells with selective medium. The selective medium kills cells that do not express the lipoprotein receptors. For example, we have used this approach to introduce functional human LDL receptors into LDL receptor-deficient Chinese hamster ovary (CHO) cell mutants. The focus of this proposal is the acetyl LDL receptor, a macrophage-specific lipoprotein receptor that mediates the endocytosis of modified lDL and the formation of foam cells in culture. CHO cells do not normally express the macrophage-specific acetyl LDL receptor. In preliminary experiments, we have used DNA transfer and lipoprotein receptor selection to isolate acetyl LDL receptor bearing CHO cells. The aim of the proposed research is to characterize in detail the lipoprotein and cholesterol metabolism of the selected cells and to compare the properties of these cells to those of true macrophages. The selected cells might be ideally suited for the somatic cell genetic analysis of macrophage-specific lipoprotein endocytosis and foam cell formation. In addition, the human acetyl LDL receptor gene will be cloned from secondary DNA-transfectants derived from these cells. Human-specific (Alu-family) repetitive DNA will be used as a probe for detecting acetyl LDL receptor genomic clones in a phage library. These genomic clones will in turn be used to isolate cDNA which will be sequenced to determine the primary structure of the acetyl LDL receptor. The methods developed to isolate CHO cells that express the acetyl LDL receptor. The methods developed to isolate CHO cells that express the acetyl LDL receptor should prove to be useful for the characterization of other tissue-specific receptors which mediate endocytosis.