THYROID HORMONE AND THE GUT

描述：(改编自调查人员摘要) 肠道的生长和分化对于维持 肠道功能正常。肠道粘膜生长和分化的紊乱 导致腹泻、吸收不良和屏障功能改变。甲状腺 激素(T3)是肠上皮细胞最有效的调节剂之一 生长和分化，但其在胃肠道的作用机制是 很大程度上是未知的。这项研究建议旨在阐明 三碘甲状腺原氨酸对胃肠道粘膜影响的分子机制 结构和功能。体内和体外研究都表明 T3对小肠上皮有两个主要作用(1) 诱导隐窝细胞增殖；(2)隐窝细胞形态改变 肠道细胞基因表达，如肠道碱性磷酸酶升高 (IAP)表达式。T3对靶组织的影响一般是 通过与相互作用的核受体蛋白结合而介导的 直接与特定的T3反应基因中的DNA顺式元件(Tre)结合。 多种形式的t3受体(Tr)由α或 βcerbA基因，包括非激素结合变异体c-erbaα-2， 它被认为可以抑制T3的作用。因为细胞的T3反应性是 根据不同RR的相对丰度，我们将在 原位杂交和隐窝-绒毛分离技术检测 小肠组织中特定细胞类型的受体表达模式 粘膜。 他们已经建立了转基因的HT-29细胞(HT-29TR)，作为一种优秀的 体外模型研究T3对肠上皮细胞的影响。 HT-29TR细胞将用T3处理，以检查生长的性质 反应，例如，~3H胸腺嘧啶核苷掺入，原癌基因诱导，以及 凝胶移位分析鉴定具有c-fox血清反应的核复合体 元素。将与特征良好的墓穴进行直接比较 细胞生长因子，EGF。 丁酸钠处理的HT-29TR细胞将被用作绒毛模型 肠上皮细胞及T3对IAP基因表达的影响。瞬变 用IAP报告基因构建物进行的转基因实验将使它们 鉴定重要的DNA顺式调控元件。最后，DNA酶1 将进行足迹和凝胶位移分析，以更准确地定义 T3调控基因背后的相关DNA-蛋白质相互作用 表情。 拟议的研究将加强对T3的作用的理解 与肠道粘膜结构和功能有关，并且应该 对发育和肿瘤的生物学有更广泛的影响。它 希望这些研究将确定潜在的治疗靶点 可在未来用于在危重期间保持肠道完整性 生病了。

DESCRIPTION: (Adapted from investigator's abstract) The processes of intestinal growth and differentiation are essential to the maintenance of normal gut function. Derangements in gut mucosal growth and differentiation result in diarrhea, malabsorption, and altered barrier function. Thyroid hormone (T3) is one of the most potent regulators of intestinal epithelial growth and differentiation, but its mechanism of action in the GI tract is largely unknown. This research proposal is designed to elucidate the molecular mechanisms by which T3 exerts its profound effects upon GI mucosal structure and function. Both in vivo and in vitro studies have demonstrated that T3 has two major effects upon the small intestinal epithelium (1) induction of crypt cell hyperplasia and (2) alteration in the pattern of enterocyte gene expression, e.g., increased intestinal alkaline phosphatase (IAP) expression. The effects of T3 on target tissues are generally mediated through binding to a nuclear receptor protein which interacts directly with DNA cis-elements (TRE) within specific T3-responsive genes. Multiple forms of the T3 receptor (TR) are encoded by either the alpha or beta cerbA genes, including a non-hormone binding variant, c-erbA alpha-2, which is thought to inhibit T3 action. Since cellular T3-responsiveness is dependent upon the relative abundance of the various TRs, we will use in situ hybridization and crypt-villus separation techniques to determine the cell type-specific patterns of TR expression within the small intestinal mucosa. They have established TR-transfected HT-29 cells (HT-29TR) as an excellent in vitro model to study the T3 effects on intestinal epithelial cells. HT-29TR cells will be treated with T3 to examine the nature of the growth response, e.g., 3H thymidine incorporation, proto-oncogene induction, and gel shift assays to identify a nuclear complex with the c-fox serum response element. Direct comparisons will be made with the well characterized crypt cell growth factor, EGF. Sodium butyrate-treated HT-29TR cells will then be used as a model of villus enterocytes and the T3 effects upon IAP gene expression examined. Transient transfection experiments with IAP reporter gene constructs will enable them to identify the important DNA cis-regulatory elements. Finally, DNAse 1 footprinting and gel shift assays will be performed to more precisely define the relevant DNA-protein interactions underlying T3-regulated gene expression. The proposed studies will enhance the understanding of the role that T3 plays in regards to intestinal mucosal structure and function, and should have broader implications for the biology of development and neoplasia. It is hoped that the studies will identify potential therapeutic targets which could be used in the future to maintain gut integrity during critical illness.