ROLE OF DIETARY CONSTITUENTS ON GENE EXPRESSION IN INTESTINAL EPITHELIUM

膳食成分对肠上皮基因表达的作用

• 批准号: 6270665
• 负责人: Ian R Sanderson
• 金额: $ 12.92万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6270665
• 关键词: MHC class II antigen; Reoviridae; antigens; dietary constituent; dietary lipid; disease /disorder model; enzyme linked immunosorbent assay; gastrointestinal epithelium; gene expression; in situ hybridization; inflammatory bowel diseases; interferon gamma; interleukin 1; interleukin 6; interleukin 8; invariant chain; laboratory mouse; macrophage inflammatory proteins; northern blottings; nutrition related tag; polymerase chain reaction; secretory immune system; short chain fatty acid; tissue /cell culture; weaning; western blottings

The mechanisms whereby chemically defined diets induce remission in Crohn's disease are not understood. Progress will depend on the examination of dietary factors in in vivo and in vitro model system where nutrients can be accurately administered and their effects measured by well- defined immunological markers. We will examine how dietary constituents influence the mucosal immune system by examining the hypothesis the dietary constituents (specifically dietary fat or antigenic load) alter the immune system of the gut by influencing the expression of genes in the epithelium. We will study the genes coding for cytokines (particularly IL-6 and IL-8-related "chemokines", IL-8 and macrophage inflammatory protein-2 [MIP-21) and the surface expression of class II major histocompatibility complexes (MHC) on the cell surfaces. We will pursue the following specific aims: Specific aim#1 Determine the influence of dietary factors on the expression IL-6, MIP-2 and class II MHC by enterocytes in vivo. We will use the differences between commercially available Mead-Johnson products to study 1) the role of fat and 2) the role of antigenic protein on the mucosal immune system. We will examine their effect on the epithelial expression of cytokines and class II MHC expression in an inflammatory bowel disease model (reovirus infected mouse) and during weaning. Our previous studies have shown that an elemental diet will alter the expression of class II MHC genes in the epithelium. We will study IL-6, MIP-2 and class II MHC and its associated invariant chain. The messenger RNAs for these molecules will be detected by RNA transfer analysis (northern blots) and by quantitative RT- PCR of RNA isolated from small intestinal epithelial cells; and by in situ hybridization of frozen intestinal sections. Proteins for class II MHC and invariant chain will be examined by Western blots. Cytokines will be studies by the generation of IL-6 and MIP-2 in freshly isolated cells using ELTSA assays. Specific aim #2: Determine the importance of nutritional factors on the expression of IL- 6, IL-8 and class IIMHC in cultured human intestinal epithelial cell lines. We will complement the in vivo studies by studying nutrients directly on the expression of cytokines and class II MHC related molecules in vitro. We will examine 1) short chain fatty acids and 2) protein and nucleotides in human intestinal epithelial cells. We will induce IL-6 and IL-8 expression by (permissive) cytokines (IL-1 and IFN-gamma) at concentrations at which expression is evident, but not maximal. We will study the effect of sodium butyrate, nucleotides and protein antigens the expression of these genes with the techniques described in specific aim #1.

化学限定饮食诱导的机制 克罗恩病的缓解尚不清楚。 进步 将取决于对饮食因素的检查 体内和体外模型系统，其中营养物质可以 准确地管理并通过良好的测量其效果 明确的免疫学标记。 我们将研究如何饮食 成分通过以下方式影响粘膜免疫系统 检查饮食成分的假设 （特别是膳食脂肪或抗原负荷）改变 通过影响肠道免疫系统的表达 上皮细胞中的基因。 我们将研究基因编码 对于细胞因子（特别是 IL-6 和 IL-8 相关的 “趋化因子”、IL-8 和巨噬细胞炎症蛋白-2 [MIP-21)和II类专业的表面表达 细胞表面的组织相容性复合物（MHC）。 我们将追求以下具体目标： 具体目标#1 确定饮食因素对 肠上皮细胞表达 IL-6、MIP-2 和 II 类 MHC 体内。 我们将利用商业之间的差异 可用美赞臣产品来研究 1) 脂肪的作用 2）抗原蛋白对粘膜免疫的作用 系统。 我们将检查它们对上皮细胞的影响 细胞因子的表达和 II 类 MHC 的表达 炎症性肠病模型（呼肠孤病毒感染小鼠） 和断奶期间。 我们之前的研究表明 基本饮食会改变 II 类 MHC 的表达 上皮细胞中的基因。 我们将研究 IL-6、MIP-2 和 II 类 MHC 及其相关的不变链。 这 这些分子的信使 RNA 将被 RNA 检测到 转移分析（Northern blots）和定量 RT- 从小肠上皮细胞中分离的 RNA 进行 PCR； 以及冷冻肠切片的原位杂交。 II 类 MHC 和不变链的蛋白质将是 通过蛋白质印迹检查。 细胞因子将被研究 在新鲜分离的细胞中产生 IL-6 和 MIP-2 使用 ELTSA 检测。 具体目标#2：确定 营养因素对IL-表达的重要性 6、培养人肠道中的IL-8和IIMHC类 上皮细胞系。 我们将补充体内 通过研究营养物质直接影响表达的研究 体外细胞因子和 II 类 MHC 相关分子。 我们 将检查 1) 短链脂肪酸和 2) 蛋白质和 人肠上皮细胞中的核苷酸。 我们将 通过（允许的）细胞因子诱导 IL-6 和 IL-8 表达 （IL-1 和 IFN-γ）表达浓度 是明显的，但不是最大的。 我们将研究效果 丁酸钠、核苷酸和蛋白质抗原 使用中描述的技术表达这些基因 具体目标#1。