TG RICH LIPOPROTEIN SYNTHESIS/SECRETION IN CACO 2 CELLS

CACO 2 细胞中富含 TG 的脂蛋白合成/分泌

基本信息

批准号：
2839027
负责人：
F JEFFREY FIELD
金额：
$ 29.42万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
财政年份：
1996
资助国家：
美国
起止时间：
1996-12-01 至 2000-11-30
项目状态：
已结题

项目摘要

DESCRIPTION (Adapted from Applicant's Abstract): The regulation of intestinal lipoprotein assembly and secretion is poorly understood. Results from our previous studies and those of others suggest that lipid availability regulates lipoprotein synthesis and secretion. Unesterified and esterified cholesterol are important components of the surface and core of the triacylglycerol-rich lipoprotein particle, respectively. The contribution of newly-synthesized cholesterol, micellar cholesterol, or plasma membrane cholesterol to the normal assembly of the lipoprotein, however, is unknown. The investigators are postulating that it is cholesterol derived from the plasma membrane which is primarily utilized for lipoprotein assembly within an intestinal cell. It is also postulated that plasma membrane cholesterol content regulates cellular cholesterol metabolism and that normal cholesterol trafficking is required for this regulation to occur. P-glycoprotein, a protein located on the plasma membrane whose function in normal intestine is unknown, is postulated to play a role in cholesterol trafficking and cholesterol metabolism. This proposal will delineate experiments which will address cholesterol trafficking in intestinal cells and define the regulation of this process. The regulation of cholesterol metabolism at the level of gene expression by changes in plasma membrane cholesterol and cholesterol trafficking will be investigated. The contribution of newly-synthesized cholesterol, micellar cholesterol, and plasma membrane cholesterol to the unesterified and esterified cholesterol content of triacylglycerol-rich lipoproteins will be studied. Regulation of lipoprotein assembly and secretion by availability of newly-synthesized cholesterol and cholesterol esters will be addressed. Lastly, the role of p-glycoprotein in cholesterol trafficking, cholesterol metabolism, and lipoprotein secretion will be studied. These experiments will be performed in CaCo-2 cells cultured on permeable supports separating an upper and lower chamber. Lipoprotein synthesis and secretion will be estimated by lipid synthesis and secretion, apoB mass secretion, synthesis and degradation of labeled apoB, and apoB mRNA abundance. The regulation of HMG-CoA reductase will be addressed by estimating reductase protein by Western blotting, mRNA abundance, and synthesis and degradation of the protein. ACAT regulation will be estimated by changes in gene expression. To address the role of p-glycoprotein in cholesterol trafficking and metabolism, the investigators will over-express p-glycoprotein in CaCo-2 cells by stable-transfection with human mdr (p-glycoprotein) cDNA and experiments are proposed in gut sacs prepared from mdr1-deficient mice.

描述（改编自申请人的摘要）：对肠道脂蛋白的组装和分泌知之甚少。结果根据我们之前的研究和其他人的研究表明，脂质可用性调节脂蛋白的合成和分泌。未酯化和酯化胆固醇是表面和核心的重要组成部分分别是富含三酰甘油的脂蛋白颗粒。这新合成的胆固醇、胶束胆固醇或质膜胆固醇到脂蛋白的正常组装，然而，未知。调查人员推测这是源自质膜的胆固醇，主要用于肠细胞内的脂蛋白组装。还假设质膜胆固醇含量调节细胞胆固醇新陈代谢和正常的胆固醇运输是必需的监管发生。 P-糖蛋白，一种位于血浆上的蛋白质膜在正常肠道中的功能尚不清楚，推测在胆固醇运输和胆固醇代谢中发挥作用。这提案将描述解决胆固醇问题的实验肠道细胞的贩运并定义了该过程的调节。在基因表达水平上调节胆固醇代谢质膜胆固醇和胆固醇运输的变化将调查了。新合成的胆固醇、胶束的贡献胆固醇和质膜胆固醇未酯化和富含三酰甘油的脂蛋白的酯化胆固醇含量为研究过。通过可用性调节脂蛋白组装和分泌新合成的胆固醇和胆固醇酯的问题将得到解决。最后，p-糖蛋白在胆固醇运输中的作用，胆固醇将研究代谢和脂蛋白分泌。这些实验将在培养于可渗透支持物上的 CaCo-2 细胞中进行，分离上室和下室。脂蛋白的合成和分泌通过脂质合成和分泌、apoB 大量分泌、合成来估计以及标记的 apoB 的降解和 apoB mRNA 丰度。的监管 HMG-CoA 还原酶将通过估计还原酶蛋白来解决蛋白质印迹、mRNA 丰度以及蛋白质的合成和降解蛋白质。 ACAT 调节将通过基因表达的变化来估计。解决 p-糖蛋白在胆固醇运输中的作用和代谢，研究人员将在 CaCo-2 中过度表达 p-糖蛋白通过用人 mdr（p-糖蛋白）cDNA 稳定转染细胞和实验是在 mdr1 缺陷小鼠制备的肠囊中进行的。