REGULATION OF HEPATIC LIPOPROTEIN SYNTHESIS

肝脏产生的脂类和载脂蛋白是 血浆中的脂蛋白水平。这项提案的总体目标是 研究肝脏合成脂蛋白的机制。这个 实现这一目标的方法将分为两个目标。 目的一是研究在大鼠肝细胞培养中的调节作用。 载脂蛋白的合成、细胞运输和分泌。监管 载脂蛋白合成的机制将通过1)杂交来测量 用载脂蛋白cDNA探针检测细胞RNA，2)免疫沉淀法 肝细胞信使核糖核酸在游离细胞内翻译所产生的前载脂蛋白 蛋白质合成系统，以及3)载脂蛋白的免疫沉淀 完整细胞经脉冲追逐标记后合成 肝细胞培养。细胞的运输和分泌将被量化 通过免疫沉淀法在亚细胞细胞匀浆中沉淀脱脂蛋白 组分，以及培养物生物合成标记后的培养基材。 细胞、亚细胞组分和培养液的载脂蛋白含量 用放射免疫测定法进行测定。肝细胞将从以下来源获得 体内载脂蛋白产生受到干扰的动物。动物 将被喂以高碳水化合物饮食、高胆固醇饮食和饮食 含有胆固醇和丙基硫氧嘧啶。这些饮食有很好的效果 确定了对血浆脂蛋白水平的影响。他们将被用来 通过使用上面的技术来量化翻译前， 肝脏调节中的翻译、翻译后事件 脂蛋白生物合成。 目的二是研究体外长期扰动对人血管内皮细胞生长的影响。 脂蛋白生物合成。为此，大鼠肝细胞已经被 通过转移SV40早期基因而永垂不朽。已经有克隆系了 可用于筛选细胞和介质中的脂质和 在确定的培养条件下确定表型 不同无性系间脂蛋白的表达不同。选定的克隆 将进行遗传和表型稳定性测试，并进行详细研究 用于载脂蛋白合成、细胞内加工和分泌。这些 实验应该促进我们对分子事件的了解 脂蛋白生物合成。

Production of lipids and apoproteins by the liver is a key determinant of lipoprotein levels in the plasma. The overall goal of this proposal is to study the mechanisms of lipoprotein biosynthesis by the liver. The approach to achieve this goal will be divided in two objectives. Objective I is to study, in rat hepatocyte cultures, the regulation of apolipoprotein synthesis, cellular transport, and secretion. Regulatory mechanisms of apoprotein synthesis will be measured by 1) hybridization assays of cellular RNA with apoprotein cDNA probes, 2) immunoprecipitation of preapoproteins produced by translation of hepatocyte mRNA in cell free protein synthesis systems, and 3) immunoprecipitation of apoproteins synthesized by intact cells subsequent to pulse chase labeling of hepatocyte cultures. Cellular transport and secretion will be quantified by immunoprecipitation of apoproteins in cell homogenates, subcellular fractions, and culture media after biosynthetic labeling of cultures. Apoprotein contents of cells, subcellular fractions, and culture media will be measured by radioimmunoassays. Hepatocytes will be obtained from animals in which apoprotein production has been perturbed in vivo. Animals will be fed a carbohydrate-rich diet, a cholesterol-rich diet, and a diet containing cholesterol and propylthiouracil. These diets have well established consequences on plasma lipoprotein levels. They will be used to quantitate, by using the above techniques, pretranslational, translational, and post-translational events in the regulation of hepatic lipoprotein biosynthesis. Objective II is to study effects of long term perturbations in vitro on lipoprotein biosynthesis. For this purpose, rat hepatocytes have been immortalized by transfer of the early genes of SV40. Clonal lines already available will be screened for cellular and media contents of lipid and apoproteins at defined culture conditions to determine whether phenotypic expression of lipoprotein production differs among clones. Selected clones will be tested for genetic and phenotypic stability and studied in detail for apoprotein synthesis, intracellular processing, and secretion. These experiments should advance our knowledge on molecular events operating in lipoprotein biosynthesis.