REGULATION OF ATROPHY-INDUCED PROGENITOR CELLS IN THE GASTRIC CORPUS

项目总结 胃上皮干细胞的特性很差，考虑到它在许多胃中的作用，这是令人惊讶的。 疾病。大剂量选择性雌激素受体调节剂三苯氧胺(HD-)杀死顶叶 细胞，并引起增殖、化生的胃反应，这种反应遵循患者的模式 慢性幽门螺杆菌感染，但在更同步和可控的模式下，具有 在注射后3天达到高峰。已发表的使用HD-的研究表明，这种反应依赖于 ℗-ERK、→、CD44、→℗-STAT3信号轴和联合增殖均源于峡部干细胞的扩张 细胞(ISC)和成熟的产酶细胞的作用，这些细胞重新编程以重新进入细胞周期，称为 招募的干细胞(RSCs)。虽然这些不断增长的人口在应对任何 化生诱因，我们最近发现高度特异的白喉毒素(DT)介导的顶叶 细胞死亡可诱导间质干细胞的扩张，但不会发生化生或RSC的生成。这两个型号现在允许我们 明确ISCs和RSCs的特征，以确定其相关的增殖机制。 我们的目标是：1)描述ISC扩张/恢复的机制，并确定CD44的具体作用 在体内以及在人和小鼠的胃器官(“胃类”)中的ISC扩张。我们也会把纯净的 DT后的ISCS和HD-后的混合ISCS+RSCs，用RNASeq分析每个群体的特征。 2)使用雷帕霉素和二甲双胍检查控制每个增殖人群的新代谢途径， 这两种药物都被认为可以减少HD-后的增殖。这些药物将在以下情况下进行体内试验 HD-和DT以及胃类中的蛋白质，以及药物筛选以确定参与其 信号通路。3)我们现在能够确定化生RSC的生成是否增加了 单独的ISC增殖增加与胃肿瘤的发生有关。我们将遵循已建立的协议，使用 监测DT或HD-周期给药时致癌物MNU对小鼠致瘤性的影响 新型近红外荧光实时成像技术无创检测肿瘤发生的全过程 在活体内发现追踪胃癌的探针。我们将追踪特定的突变和必需的蛋白质 癌症进展和关注我们庞大的人类胃癌组织生物库中发现的任何候选组织。 我们的DT和HD-模型首次允许我们分离胃内的间质干细胞和系膜细胞的增殖， 最后，考虑到每个人口的详细特征。我们的实验旨在利用这个机会 将我们已知的增殖轴更明确地归因于ISC和/或RSC的扩张，定义了新的代谢 参与控制每个种群中的扩散的途径，并运行无偏见的筛查以确定其他 这些通路的调节器。最后，我们的模型允许我们测试一种新的理论，即固体组织癌如何 研究成熟的有丝分裂后细胞(RSC)的几轮重新编程和细胞周期重新进入 比起单纯的ISC增殖增加，肿瘤发生的风险更大。

PROJECT SUMMARY The epithelial stem cell of the stomach is poorly characterized, which is surprising given its role in many gastric diseases. High doses of the Selective Estrogen Receptor Modulator (SERM) tamoxifen (HD-Tam) kill parietal cells and causes a proliferative, metaplastic gastric response which follows the pattern seen in patients chronically infected with Helicobacter pylori, but in a more synchronous and controllable model, with the effect peaking at 3 days post injections. Published studies using HD-Tam show that this response is dependent on a ℗-ERK→CD44→℗-STAT3 signaling axis and combines proliferation both from expansion of the isthmal stem cell (iSC) and contribution from mature zymogenic cells which reprogram to re-enter the cell cycle, termed recruited stem cells (rSCs). While these proliferating populations are inseparable in their response to any metaplasia-causing agent, we recently discovered that highly specific diphtheria toxin (DT)-mediated parietal cell death induces iSC expansion but no metaplasia or rSC generation. These two models now allow us to specifically characterize iSCs and rSCs to determine their relative mechanisms of proliferation. Our Aims are: 1) Characterize mechanisms of iSC expansion/recovery and determine the specific role of CD44 in iSC expansion both in vivo and in human and murine gastric organoids (“gastroids”). We will also sort pure iSCs following DT and mixed iSCs+rSCs following HD-Tam and run RNA-Seq to characterize each population. 2) Examine new metabolic pathways governing each proliferative population using rapamycin and metformin, both of which are seen to decrease proliferation following HD-Tam. These drugs will be tested in vivo following HD-Tam and DT as well as in gastroids, along with a drug screen to identify other proteins involved in their signaling pathways. 3) We are now able to determine whether metaplastic rSC generation increases risk of gastric tumorigenesis over increased iSC proliferation alone. We will follow an established protocol using the carcinogen MNU to drive tumorigenesis in mice while treating with cycles of DT or HD-Tam while monitoring the entire course of tumorigenesis non-invasively with Near Infrared Fluorescence live imaging with novel probes found to track gastric cancer in vivo. We will track specific mutations and proteins necessary for this cancer progression and focus on any candidates found with our large biobank of human gastric cancer tissues. Our DT and HD-Tam models allow us to separate iSC and rSC proliferation in the stomach for the first time, finally allowing for detailed characterization of each population. Our experiments aim to use this opportunity to attribute our known proliferative axis more specifically to iSC and/or rSC expansion, define new metabolic pathways involved in governing proliferation in each population, and run unbiased screens to identify additional modulators of these pathways. Finally, our models allow us to test a new theory for how solid tissue cancers arise, investigating whether rounds of reprogramming and cell cycle re-entry of mature, post-mitotic cells (rSC generation) increase risk for tumorigenesis over increased iSC proliferation alone.