NUTRITIONAL REGULATION OF APOPROTEIN B GENE EXPRESSION

建议研究载脂蛋白的营养调节 B(ApoB)基因表达。这项提案的总体目标是界定 载脂蛋白B基因调控的分子机制(S) 胆固醇和脂肪酸在HepG2和Caco-2细胞中的表达 分别代表人类的肝脏和肠道。重点放在 这一建议的目的是确定调节合成的因素 和翻译后水平的载脂蛋白B的分泌。的具体目标 这项建议是：1)研究油酸盐和胆固醇的影响 关于载脂蛋白B的胞内降解。2)证实…的存在 代谢不同的ApoB胞内池及其比率 营业额受脂肪酸和胆固醇的影响。3)确定 胆固醇酯合成在酰基-2，4-二苯基-2，4，4，4，4，6-二苯基-4，4，4，4，4，4-三甲基-4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4， 辅酶A：胆固醇酰基转移酶(ACAT)调节载脂蛋白B合成和 分泌物。4)检查可能存在的可能存在的因素 在转录水平调节载脂蛋白B基因的表达，尤其是， 转录后信使核糖核酸稳定性水平。为了实现这一目标， HepG2和Caco-2细胞条件培养液中载脂蛋白B的浓度 分别与胆固醇和油酸盐孵育，将通过 电免疫分析、微量ELISA法和单特异性免疫印迹 抗载脂蛋白B的多克隆或单克隆抗体。可能产生的影响 胆固醇和油酸对Caco-2细胞载脂蛋白B mRNA编辑的影响 条件培养基在梯度十二烷基硫酸钠上的电泳法测定 聚丙烯酰胺凝胶与特异性单抗的免疫印迹 ApoB的N-末端结构域和C-末端结构域。胞内率 载脂蛋白B的降解、DISTINCT的存在和周转率 ApoB的胞内池及其ACAT的调节作用 过程将通过脉冲追逐研究和亚细胞来确定 分级后免疫沉淀，电泳法， 免疫印迹和/或放射自显影结合使用 特异性蛋白水解酶、ACAT抑制剂和高分辨电子 显微免疫细胞化学。阐明分子机制(S) 参与脂肪酸对载脂蛋白B基因表达的调节 胆固醇，细胞信使核糖核酸测定，信使核糖核酸稳定性测定，凝胶 迁移迟缓和/或核启动试验和瞬时表达 将在HepG2和Caco-2细胞中应用ApoB/CAT杂合构建。 从这些研究中获得的信息可能被用于一种新的方法 治疗和控制高脂蛋白血症和动脉粥样硬化。

Research is proposed to study the nutritional regulation of apolipoprotein B (ApoB) gene expression. The overall goal of this proposal is to define the molecular mechanism(s) involved in the regulation of ApoB gene expression by cholesterol and fatty acids in HepG2 and Caco-2 cells representing human liver and intestine, respectively. The major emphasis of this proposal is to identify the factors which regulate the synthesis and secretion of ApoB at the posttranslational level. The specific aims of this proposal are: 1) To investigate the effect of oleate and cholesterol on the intracellular degradation of ApoB. 2) To establish the existence of metabolically distinct intracellular pools of ApoB and their rate of turnover as influenced by fatty acids and cholesterol. 3) To determine the role of cholesteryl ester synthesis through the activation of acyl- CoA:cholesterol acyltransferase (ACAT) in regulation of ApoB synthesis and secretion. 4) To examine the possible existence of factors which might regulate ApoB gene expression at transcriptional and, in particular, posttranscriptional mRNA stability levels. To achieve this goal, the concentration of ApoB in the conditioned media of HepG2 and Caco-2 cells incubated with cholesterol and oleate, respectively, will be determined by electroimmunoassay, microELISA and immunoblotting using monospecific polyclonal or monoclonal antibodies to ApoB. The potential effects of cholesterol and oleate on editing of ApoB mRNA in Caco-2 cells will be determined by electrophoresis of conditioned medium on gradient SDS polyacrylamide gels and immuno-blotting with monoclonal antibodies specific for N- and C-terminal domains of ApoB. The rate of intracellular degradation of ApoB, the existence and the turnover rates of distinct intracellular pools of ApoB and the regulatory role of ACAT in these processes will be determined by pulse-chase studies and subcellular fractionation followed by immunoprecipitation, electrophoresis, immunoblotting and/or autoradiography in conjunction with the use of specific proteases, inhibitors of ACAT and high resolution electron microscopic immunocytochemistry. To elucidate the molecular mechanism(s) involved in the regulation of ApoB gene expression by fatty acids and cholesterol, cellular mRNA measurement, mRNA stability determination, gel migration retardation and/or nuclear run-on assay, and transient expression of hybrid ApoB/CAT construct in HepG2 and Caco-2 cells will be employed. Information obtained from these studies may be used for a new approach to treatment and control of hyperlipoproteinemias and atherosclerosis.