NUTRITIONAL REGULATION OF APOPROTEIN B GENE EXPRESSION

因此，研究载脂蛋白的营养调节作用是必要的 B（ApoB）基因表达。 本提案的总体目标是确定 ApoB基因调控的分子机制 胆固醇和脂肪酸在HepG 2和Caco-2细胞中的表达 分别代表人的肝脏和肠。 主要重点 这一建议的目的是确定调节合成的因素， 和ApoB在翻译后水平的分泌。 的具体目标 本研究的主要目的是：1）研究油酸和胆固醇对大鼠肝纤维化的影响 对ApoB细胞内降解的影响 2)证实存在 代谢不同的ApoB细胞内池及其代谢率 周转率受脂肪酸和胆固醇影响。 3)确定 胆固醇酯合成的作用，通过激活酰基- 辅酶A：胆固醇酰基转移酶（ACAT）在ApoB合成和 分泌物 4)研究可能存在的因素， 在转录水平调节ApoB基因表达，特别是， 转录后mRNA稳定性水平。 为了实现这一目标， HepG 2和Caco-2细胞条件培养基中ApoB的浓度 分别与胆固醇和油酸盐孵育，将通过 电免疫测定、微量ELISA和免疫印迹， ApoB的多克隆或单克隆抗体。 的潜在影响 胆固醇和油酸对Caco-2细胞中ApoB mRNA编辑的影响， 通过条件培养基在梯度SDS上电泳测定 聚丙烯酰胺凝胶和单克隆抗体免疫印迹 ApoB的N-和C-末端结构域。 细胞内 ApoB的降解，不同的ApoB的存在和周转率 ApoB的细胞内池和ACAT在这些中的调节作用 过程将通过脉冲追踪研究和亚细胞 分级分离，然后免疫沉淀，电泳， 免疫印迹和/或放射自显影结合使用 特异性蛋白酶、ACAT抑制剂和高分辨电子 显微免疫细胞化学 阐明分子机制 参与脂肪酸对ApoB基因表达的调节， 胆固醇，细胞mRNA测量，mRNA稳定性测定，凝胶 迁移阻滞和/或核运行试验，以及瞬时表达 将使用在HepG 2和Caco-2细胞中的杂合ApoB/CAT构建体。 从这些研究中获得的信息可用于一种新的方法， 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.