CHARACTERIZING MURINE GI STEM CELLS: NEW MOLECULAR TOOLS

描述(申请人提供)：干细胞(Sc)几乎肯定是肠癌的起源细胞。但是，sc机制尚不清楚，因为目前还没有识别sc的特异标记物。我们的长期目标是使用动物模型来开发和测试用于在活体内识别成人肠道干细胞的研究工具。在这里，我们将测试一种工具，GFP-TCF4转基因小鼠。我们设计了这个小鼠系，当TCF4被激活时，显示绿色荧光蛋白阳性(GFP+)细胞。TCF4之所以重要，是因为1)由于致癌的APC突变，肠道肿瘤激活了TCF4；2)现有证据表明，正常隐窝中的TCF4激活是sc所特有的。因此，TCF4激活的标志物可能是SC的标志物。我们让GFP-TCF4小鼠与ApcMin小鼠杂交，因为后者患肠癌的风险很高&评估APC突变对TCF4激活的影响。目的1：鉴定、分离和鉴定GFP-TCF4小鼠GFP+肠上皮细胞。这些小鼠中的GFP+肠细胞似乎局限于隐窝底部(SC所在的地方)。为了量化这一发现，我们将a)使用荧光显微镜/先进的图像分析(肠道切片)测量体内沿隐窝轴线的GFP信号强度，以及b)流式细胞术分选(分离的固定的、GFP+的分离的来自纯化的整个隐窝的肠细胞)。假设1(H1)：GFP+的肠上皮细胞仅位于隐窝底部，是一小部分隐窝细胞，染色为核(-catenin)((C)。目的2.使用上述显微镜和细胞术，观察GFP报告基因是否对TCF4激活的变化做出反应。H2：GFP+的隐窝细胞数为核(c定位在突变隐窝中增加(正常粘膜[1个APC突变]，腺瘤性粘膜[2个突变])。目的3.分离GFP+和GFP-肠上皮细胞。在这里，我们将分离存活的(非固定的)肠上皮细胞，并用流式细胞仪对它们进行分类。H3：防止肠细胞单个细胞悬液中的结块将允许对活细胞进行流式细胞仪分选；GFP+但不是GFP-细胞将显示核(c染色和Hoechst染料外流检测阳性，这是一种功能性SC特性)。意义重大。如果预测成立，Aim 1的结果将表明GFP表达ID的肠道SC(或sc样细胞)；Aim 2的结果，记者对TCF4激活的变化做出反应；Aim 3的结果，活的GFP+肠道细胞是可分离的和可鉴定的，因此可以测试它们是否具有其他sc特性。然后，人们可以评估SC在肠道肿瘤的启动和促进中的作用。

DESCRIPTION (provided by applicant): Stem cells (sc) are almost certainly the cells of origin of intestinal cancer. But, sc mechanisms are obscure because there are no specific markers to identify SO. Our long-term objective is to use animal models to develop & test research tools for identifying adult intestinal sc in vivo. Here we will test one tool, GFP-Tcf4 transgenic mice. We designed this mouse line to display green fluorescence protein positive (GFP+) cells when Tcf4 is activated. Tcf4 is important because 1) intestinal tumors have activated Tcf4 due to cancer-causing Apc mutations & 2) available evidence suggests that Tcf4 activation in normal crypts is unique to sc. Thus, a marker for Tcf4 activation may be a marker for sc. We crossed GFP-Tcf4 mice with ApcMin mice because the latter are at high risk for intestinal cancer & to assess effects of Apc mutations on Tcf4 activation. AIM 1: To identify (ID), isolate & characterize GFP+ enterocytes from GFP-Tcf4 mice. GFP+ enterocytes in these mice appear localized to the crypt base (where SC reside). To quantify this finding, we will a) use fluorescence microscopy/advanced image analysis (of intestinal sections) measuring in vivo GFP signal intensity along the crypt axis, & b) flow cytometry sorting (of isolated fixed, GFP+ enterocytes derived from purified whole crypts). Hypothesis 1(H1): GFP+ enterocytes are only in the crypt base, are a small % of crypt cells, stain for nuclear (-catenin ((c). AIM 2. To see, using microscopy & cytometry as above, if the GFP reporter responds to changes in Tcf4 activation. H2: The # of crypt cells that are GFP+ & show nuclear (c localization are increased in mutant crypts (normal-appearing mucosa [1 Apc mutation], adenomatous mucosa [2 mutations]). AIM 3. To isolate viable GFP+ & GFP- enterocytes. Here we will isolate viable (not fixed) enterocytes & sort them by flow cytometry. H3: Prevention of clumping in single cell suspensions of enterocytes will permit flow cytometric sorting of viable cells; GFP+ but not GFP- cells will show nuclear (c staining & test positive for Hoechst dye efflux, a functional SC property. Significance. If predictions hold, Aim 1 results will suggest that GFP expression ID's intestinal SC (or sc-like cells); Aim 2 results, that the reporter responds to changes in Tcf4 activation; Aim 3 results, that viable GFP+ intestinal cells are isolatable and characterizable hence can be tested to see if they have other sc properties. One could then evaluate the role of SC in initiation & promotion of intestinal neoplasms.