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MOLECULAR MECHANISMS OF INTESTINAL ATROPHY/HYPERPLASIA

肠萎缩/增生的分子机制

基本信息

批准号：
2749502
负责人：
RICHARD A. HODIN
金额：
$ 8.37万
依托单位：
BETH ISRAEL DEACONESS MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-08-01 至 1999-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2749502
关键词：
DNA footprinting alkaline phosphatase alternatives to animals in research atrophy cell differentiation cell growth regulation gastrointestinal epithelium gel mobility shift assay gene expression genetic regulatory element human genetic material tag hyperplasia intestinal villi molecular pathology tissue /cell culture transfection

项目摘要

The mammalian small intestine is lined by a continuously renewable population of simple columnar cells which arise in the crypts and acquire a differentiated phenotype as they migrate to the villus compartment. The enterocytes, which comprise 95% of the differentiated villus cells, possess a brush-border at their luminal surface containing the enzymes and transporters essential for the digestion and absorption of dietary nutrients. Villus atrophy occurs in a variety of disease states and is associated with diarrhea, malabsorption, and impairment in gut barrier function. An understanding of the molecular mechanisms underlying Villus atrophy could lead to important therapies designed to maintain both the structural and functional integrity of the gastrointestinal tract. It has generally been assumed that the physiological derangements associated with villus atrophy are due to a decrease in the number of absorptive epithelial cells; that is, the enterocytes are unchanged, there are just less of them. However, data from four independent rat models of villus atrophy/hyperplasia demonstrate that enterocyte phenotype changes dramatically as a function of epithelial growth state. It is clear from these preliminary studies that the process of crypt-villus differentiation is closely linked to the proliferative state of the epithelium. Intestinal alkaline phosphatase (IAP) is a brush-border enzyme expressed exclusively in differentiated enterocytes. IAP is unique among the various markers of enterocyte differentiation in that its expression is regulated by developmental, dietary, and hormonal factors in vivo, and that it undergoes regulated expression in vitro (HT-29 cells). Thus, IAP will be used as a tool to elucidate the molecular mechanisms underlying two distinct but related processes: (1) the crypt-villus axis of enterocyte differentiation, and (2) the alterations in enterocyte phenotype that accompany changes in epithelial growth state. Functional studies on the endogenous IAP gene, as well as transfection experiments using IAP reporter constructs, will allow for the identification of those elements within the IAP gene that are transcriptionally active and which mediate its regulation within the contexts of both enterocyte growth and differentiation. DNase l footprinting and mobility shift assays will then be performed in order to identify and characterize the specific sites of DNA-protein interactions which are responsible for IAP regulation. Finally, we plan to identify new genes whose expression is altered as a function of intestinal atrophy/hyperplasia, to more fully understand the genetic programs which underlie the changes in enterocyte phenotype. These studies will bring together the molecular mechanisms which regulate the crypt-villus axis of differentiation with those regulating intestinal epithelial proliferation, and, as such, could lead to a unifying model with which to understand the processes of enterocyte growth and differentiation.

哺乳动物的小肠由一层不断更新的一群简单的柱状细胞，出现在隐窝中，当它们迁移到绒毛隔室时分化的表型。的肠上皮细胞，占分化绒毛细胞的95%，在其含有酶的管腔表面具有刷状缘，食物消化和吸收所必需的转运蛋白营养素绒毛萎缩发生在多种疾病状态中，与腹泻、吸收不良和肠道屏障受损相关功能了解绒毛的分子机制萎缩可能会导致重要的治疗方法，旨在维持这两个胃肠道的结构和功能完整性。人们普遍认为，与绒毛萎缩相关的是由于绒毛数量减少，吸收上皮细胞;也就是说，肠上皮细胞是不变的，只是数量少而已然而，来自四个独立的大鼠模型的数据，绒毛萎缩/增生表明肠上皮细胞表型改变显著地作为上皮生长状态的函数。清楚地表明这些初步研究表明，与上皮细胞的增殖状态密切相关。肠碱性磷酸酶（IAP）是一种刷状缘酶，只在分化的肠上皮细胞中。IAP在各种肠上皮细胞分化的标志物，因为其表达受到调节受体内发育、饮食和激素因素的影响，在体外（HT-29细胞）进行调节表达。因此，IAP将作为一种工具来阐明两个潜在的分子机制，两个不同但相关的过程：（1）肠上皮细胞的隐窝绒毛轴分化，和（2）肠上皮细胞表型的改变，伴随上皮生长状态的变化。内源性IAP基因的功能研究以及转染使用IAP报告构建体的实验，将允许识别IAP基因内的那些元件，转录活性，并介导其调节内肠上皮细胞生长和分化的背景。脱氧核糖核酸酶1 然后将进行足迹法和迁移率变化测定，识别和表征DNA-蛋白质相互作用的特定位点负责IAP监管。最后，我们计划确定新的基因的表达被改变作为一个功能的肠萎缩/增生，以更充分地了解遗传程序，是肠上皮细胞表型变化的基础。这些研究将汇集调节细胞凋亡的分子机制，隐窝-绒毛分化轴与调节肠上皮细胞增殖，因此，可能导致一个统一的模型，了解肠上皮细胞生长的过程，分化