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)的群体。发展鉴定结肠隐窝中的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。我们的近期目标是开发结肠干细胞富集型(SCE)制备的标记物。基于阵列的基因表达谱将被用于根据SC的解剖和功能特性(位于隐窝底部的位置和克隆性)来表征所制备的结肠姐妹染色单体交换。我们将研究从纯化的正常隐窝(目标1)和含有突变的APC的隐窝(来自家族性腺瘤性息肉病[FAP]患者；目标2)制备的姐妹染色单体交换，以比较它们的基因表达谱。我们将检验两个假设：H1：在隐窝基础细胞中选择性表达的基因(相对于整个隐窝)也选择性地在克隆细胞中表达(相对于整个隐窝)。H2：与正常隐窝相比，在隐窝基质细胞和/或克隆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标本的组织样本，提纯结肠隐窝，并进行微阵列分析以确定姐妹染色单体交换准备的特征。目的1：测定正常人隐窝制备的姐妹染色单体交换的基因表达谱。任务1.A：评估从纯化的隐窝底部制备姐妹染色单体交换的微阵列图谱。任务1.b评估从软琼脂集落中克隆的隐窝细胞制备姐妹染色单体交换的微阵列特征。任务1.c确定I.a和1.b的图谱中共有哪些基因，并建立目标阵列。目的：研究异常隐窝中姐妹染色单体交换标记基因的表达模式和水平。任务2：使用靶向阵列(1.c)比较FAP和正常人纯化的整个隐窝中的基因表达水平和模式。我们预测：i)每个姐妹染色单体交换准备(基础细胞和克隆细胞)都有一个独特的基因表达模式，以及ii)这些模式是相似的。我们还预测，与正常隐窝相比，姐妹染色单体交换准备的遗传标记在FAP隐窝中的表达水平有所增加。我们的结果将提供基因表达谱，可能作为结肠干细胞群体的特异性标记，或者至少作为姐妹染色单体交换准备的标记。有了姐妹染色单体交换准备的标记，未来的研究可能会通过各种实验方法导致结肠干细胞的特定标记。有了SC的特定标记物，我们可以直接测试结肠癌发生是因为APC突变导致SC过度生产的想法。

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.