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。发展在结肠隐窝中识别SC的能力将使研究它们在结肠癌起源中的假定作用成为可能。我们的直接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的解剖和功能特性（位于隐窝底部和克隆原性）制作的结肠SCE制剂。我们将研究从纯化的正常隐窝（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）中制备的SCE，比较它们的基因表达谱。我们将验证两个假设：H1：基因在隐窝基底细胞中选择性表达（与整个隐窝相比），也在克隆源细胞中选择性表达（与整个隐窝相比）。H2：与正常隐窝相比，在隐窝基部细胞和/或克隆原细胞中选择性表达的基因在FAP隐窝中的表达增加。将收集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制剂。目的1：确定正常人隐窝SCE制剂的基因表达谱。任务1。a：从纯化的隐窝底部评估SCE制备的微阵列谱。任务1。b .评估软琼脂糖菌落中隐窝克隆生成细胞制备SCE的微阵列谱。任务1.c确定哪些基因在I.a和I.a中都是共同的。B并构建目标数组。目的2：确定异常隐窝（FAP隐窝）中SCE标记基因的基因表达模式和水平。任务2：使用目标阵列比较FAP纯化的整个隐窝与正常个体的基因表达水平和模式（1.1）。我们预测：i)每种SCE制剂（碱基细胞和克隆细胞）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，与正常隐窝相比，SCE制剂的遗传标记物在FAP隐窝中的表达水平增加。我们的结果将提供基因表达谱，可能作为结肠SC群体或至少是SCE制剂的特定标记。有了SCE制剂的标记物，未来的研究可以通过各种实验方法，找到结肠SC的特异性标记物。有了SC的特异性标记物，我们可以直接验证结肠癌发生的原因是APC突变导致SC过量产生的观点。

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.