MARKERS FOR STEM CELL-LIKE COLONIC CRYPT BASE CELLS

描述（由申请人提供）：我们的长期目标是鉴定和表征肠道干细胞（SC）群体。 发展鉴定结肠隐窝中SC的能力将使研究其在结肠癌起源中的假定作用成为可能。 我们的近期目标是开发结肠干细胞富集（SCE）制剂的标记物。 基于阵列的基因表达谱将用于表征基于SC的解剖学和功能特性（位于隐窝底部和克隆形成）制备的结肠SCE制备物。 我们将研究SCE制剂从纯化的正常隐窝（目的1）和含有突变APC的隐窝（来自家族性腺瘤性息肉病[FAP]患者;目的2），以比较它们的基因表达谱。我们将测试两个假设：H1：在隐窝基底细胞中选择性表达的基因（与整个隐窝相比）也在克隆形成细胞中选择性表达（与整个隐窝相比）。H2：与正常隐窝相比，在隐窝基底细胞和/或克隆形成细胞中选择性表达的基因在FAP隐窝中的表达增加。 将采集来自手术结肠切除术标本的组织样本，纯化结肠隐窝，并进行微阵列分析以表征SCE制备物。 目的1：确定正常人隐窝SCE制备物的基因表达谱。 任务1.a：评价纯化隐窝底部SCE制备物的微阵列图谱。 任务1.b评价软琼脂中隐窝克隆形成细胞的SCE制备的微阵列图谱。 任务1.c确定哪些基因是I.a和1.b中的谱所共有的，并构建靶向阵列。目的2：探讨异常隐窝（FAP隐窝）中SCE标记基因的表达模式和水平. 任务2：使用靶向阵列比较来自FAP与正常个体的纯化的完整隐窝中的基因表达水平和模式（1.c）。 我们预测：i）每个SCE制备物（基础细胞和克隆形成细胞）都有独特的基因表达模式，ii）这些模式是相似的。 我们还预测，SCE制剂的遗传标记在FAP隐窝中的表达水平比正常隐窝增加。 我们的研究结果将提供基因表达谱，可能作为结肠SC人群或至少SCE制剂的特异性标志物。 随着SCE制剂的标记，未来的研究可能会导致，通过各种实验方法，结肠SC的特异性标记。与SC的特异性标记，我们可以直接测试的想法，结肠癌的发生，因为APC突变导致SC生产过剩。

DESCRIPTION (provided by applicant): Our long-term objective is to identify and characterize populations of intestinal stem cells (SC). Developing the ability to identity SC in colonic crypts will make possible investigation into their putative role in the origin of colon cancer. Our immediate objective is to develop markers for colonic stem cell enriched (SCE) preparations. Array-based gene expression profiling will be used to characterize colonic SCE preparations made based on anatomical and functional properties of SC (location at the bottom of the crypt & clonogenicity). We will investigate SCE preparations from purified normal crypts (Aim 1) & from crypts that contain mutant APC (from familial adenomatous polyposis [FAP] patients; Aim 2) to compare their gene expression profiles. We will test two hypothesis: H1: Genes selectively expressed in crypt base cells (compared to the whole crypt) are also selectively expressed in clonogenic cells (compared to the whole crypt). H2: Genes selectively expressed in crypt base cells and/or clonogenic cells have increased expression in FAP crypts compared to normal crypts. Tissue samples from surgical colectomy specimens will be collected, colonic crypts purified, and microarray analysis done to characterize SCE preparations. AIM 1: To determine gene expression profiles for SCE preparations from normal human crypts. Task 1.a: evaluate microarray profiles for SCE preparations from the bottom of purified crypts. Task 1.b evaluate microarray profiles for SCE preparations from crypt clonogenic cells from colonies in soft agarose. Task 1.c determine which genes are common to profiles in both I.a and 1.b and build a targeted array. AIM 2: To determine gene expression patterns and levels for SCE marker genes in abnormal crypts, (FAP crypts). Task 2: compare gene expression levels and patterns in purified whole crypts from FAP vs. normal individuals using the targeted array (1 .c). We predict: i) there is a unique gene expression pattern for each SCE preparation (base & clonogenic cells), and ii) these patterns are similar. We also predict that genetic markers for SCE preparations have increased levels of expression in FAP crypts compared to normal crypts. Our results will provide gene expression profiles that might serve as specific markers for colonic SC populations or at least for SCE preparations. With markers for SCE preparations, future research could lead, through various experimental approaches, to specific markers for colonic SC. With specific markers for SC, we could directly test the idea that colon cancer initiation occurs because APC mutation leads to SC overproduction.