Molecular analysis of intestinal stem cells in the context of premature aging, chronic inflammation and colorectal carcinogenesis

结直肠癌是工业化国家最常见的肿瘤之一，发病率和死亡率都很高。特别是老年患者和患有炎症性肠病的患者患结直肠癌的风险增加。作为肿瘤发生的细胞起源，肠道干细胞表达所谓的富含亮氨酸的重复g蛋白偶联受体5 （Lgr5）最近成为科学关注的焦点。衰老和慢性炎症对肠道干细胞的影响在很大程度上仍是未知的，将在本研究项目中进行研究。慢性炎症和早衰动物模型将与Lgr5模型相结合。端粒酶缺陷的动物端粒功能失调将作为衰老模型。将化学诱导的慢性结肠炎（DSS colitis）动物作为炎症模型。从这些模型动物和各自的对照组中分离出lgr5阳性肠道干细胞。然后，该项目将分为三个部分：(1)将使用下一代测序（NGS）分析分离的肠道干细胞，以确定肿瘤相关信号通路的遗传差异。在这项检查中，将比较过早衰老的、慢性炎症的和健康的干细胞。NGS分析将用于确定候选基因，这些基因对肠道干细胞的生长、存活和增殖有积极或消极的影响。（II）下一步将是为这些候选基因创建特异性shrna，并将在体外检测其对lgr5阳性肠道干细胞的影响。基因敲低导致体外肠道干细胞的生长和存活增加，将用于生产转基因肠道类器官。（三）将这些肠道类器官移植到健康动物体内，研究衰老和慢性炎症对体内植入、分化和结直肠癌发生的影响。

Colorectal cancer is one of the most common tumors in industrialized countries and associated with a high morbidity and mortality. Especially elderly patients and patients with inflammatory bowel disease have an increased risk to develop colorectal cancer. As cellular origin of tumorigenesis intestinal stem cells that express the so-called leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) recently moved into the scientific focus. The effects of aging and chronic inflammation on intestinal stem cells are still largely unknown and will be investigated in this research project.Animal models of chronic inflammation and premature aging will be combined with an Lgr5 model. Telomerase-deficient animals with dysfunctional telomeres will serve as an aging model. Animals after chemically-induced chronic colitis (DSS colitis) will serve as an inflammation model. Lgr5-positive intestinal stem cells are isolated from the animals of these models and respective control groups. Then the project will be divided into three parts: (I) The isolated intestinal stem cells, will be analyzed using next-generation sequencing (NGS) to identify genetic differences in tumor-associated signaling pathways. In this examination, prematurely-aged, chronically-inflamed and healthy stem cells will be compared. NGS analysis will be used to identify candidate genes, which have a positive or negative effect on growth, survival and proliferation of intestinal stem cells. (II) The next step will be the creation of specific shRNAs for these candidate genes and their effect on Lgr5-positive intestinal stem cells will be examined in vitro. Gene knockdowns, which lead to an increase in growth and survival of intestinal stem cells in vitro, will be used to produce genetically modified intestinal organoids. (III) These intestinal organoids will be transplanted into healthy animals in order to investigate the influence of aging and chronic inflammation on engraftment, differentiation and colorectal carcinogenesis in vivo.