Aging Intestinal Stem Cells and Insulin/IGF System

描述（由申请人提供）：肠上皮干细胞（IESC）在整个生命周期中更新小肠和结肠上皮，并且对于损伤后的上皮修复和再生至关重要。与衰老相关的 IESC 变化以及更新或修复肠上皮的能力可能会导致感染、损害消化和吸收能力、影响结直肠癌的易感性并限制对化疗或放疗的耐受能力。由于缺乏有效的 IESC 生物标志物，对年龄引起的 IESC 变化的理解受到了阻碍。该提案将使用 Sox9-EGFP 报告小鼠，其中不同水平的 Sox9-EGFP 表达允许直接可视化和分离 IESC、祖细胞、肠内分泌细胞 (EEC) 和其他分化的肠上皮细胞。使用 Sox9-EGFP 小鼠，我们获得了与衰老相关的 IESC 扩张的直接证据，并且胰岛素样生长因子 1 (IGF1) 有效且特异性地促进年轻人受伤后 IESC 的再生。 IGF1、IGF2 或胰岛素的水平深刻影响寿命，并可能决定用于组织修复和完整性的多能或组织限制性干细胞的存活和数量。 IGF 和胰岛素信号通过 IGF1 受体 (IGF1R) 或胰岛素受体 (IR) 产生。 IR 以 IR-B 亚型存在，介导胰岛素的代谢作用，而 IR-A 亚型可能与生长特别相关。初步数据表明 IGF1R、IR-A 和 IR-B 的表达模式预测了这些受体在 IESC、祖细胞、EEC 和其他分化的肠上皮谱系中的不同作用。肠上皮中具有完全 Villin-Cre (VC) 介导的缺失 IR (VC-IR¿/¿) 或 IGF1R (VC-IGF1R ¿/¿) 的小鼠与 Sox9-EGFP 小鼠杂交，将用于测试一个中心假设，即 IR 通过限制 IGF1R 的促有丝分裂抗凋亡作用和促进维持的分化功能，保护 IESC 和肠上皮免受衰老诱导的功能障碍。 目标#1将定义衰老对IESC、祖细胞、EEC或分化谱系的数量、功能和转录组的影响，以及这些老化细胞在损伤后再生的能力目标#2将定义IR缺失对IESC和祖细胞、EEC、分化谱系以及在整个正常衰老过程中或在IESC和损伤后上皮再生期间对胰岛素或IGF的反应的影响目标#3将定义IGF1R删除对IESC和损伤后的上皮再生的影响祖细胞、EEC、分化谱系以及在整个正常衰老过程中或在 IESC 和损伤后上皮再生期间对胰岛素或 IGF 的反应。研究结果将从根本上增进我们对衰老对 IESC 影响的认识，并确定新的机制、生物标志物和潜在的治疗靶点，以更好地监测和促进成功的 IESC 和肠道衰老。

DESCRIPTION (provided by applicant): Intestinal epithelial stem cells (IESC) renew the small intestine and colon epithelium throughout life, and are critical to epithelial repair and regeneration after injury. Aging-associated alterations in IESC and ability to renew or repair the intestinal epithelium may predispose to infection, impair digestive and absorptive capabilities, impact susceptibility to colorectal cancer and limit ability to tolerate chemo- or radiation therap. Understanding of age-induced changes in IESC has been hampered by lack of valid IESC biomarkers. This proposal will use a Sox9-EGFP reporter mouse, where distinct levels of Sox9-EGFP expression permit direct visualization and isolation of IESC, progenitors, enteroendocrine cells (EEC) and other differentiated intestinal epithelial cells. Using Sox9-EGFP mice we have direct evidence for aging-associated IESC expansion and that insulin-like growth factor 1 (IGF1) potently and specifically promotes IESC regeneration after injury in young adults. Levels of IGF1, IGF2 or insulin profoundly impact lifespan, and may dictate the survival and numbers of pluripotent or tissue-restricted stem cells for tissue repair and integrity. IGFs and insulin signa through the IGF1 receptor (IGF1R) or the insulin receptor (IR). IR exists as an IR-B isoform, which mediates metabolic effects of insulin and an IR-A isoform that may be particularly relevant to growth. Preliminary data demonstrate expression patterns of IGF1R, IR-A and IR-B which predict distinct roles of these receptors in IESC, progenitors, EEC and other differentiated intestinal epithelial lineages. Mice with complete Villin-Cre (VC)- mediated deletion IR (VC-IR¿/¿) or IGF1R (VC-IGF1R¿/¿) in intestinal epithelium, cross-bred with Sox9-EGFP mice, will be used test a central hypothesis that IR protects IESC and intestinal epithelium from aging- induced dysfunction by limiting mitogenic anti-apoptotic actions of IGF1R and promoting maintained differentiated function. Aim #1 will define the impact of aging on number, function and transcriptome of IESC, progenitors, EEC or differentiated lineages, and the ability of these aged cells to regenerate after injury Aim #2 will define the impact of IR deletion on IESC and progenitors, EEC, differentiated lineages and response to insulin or IGFs throughout normal aging, or during IESC and epithelial regeneration after injury Aim#3 will define the impact of IGF1R deletion on IESC and progenitors, EEC, differentiated lineages and response to insulin or IGFs throughout normal aging, or during IESC and epithelial regeneration after injury. Findings will fundamentally advance our knowledge of the effects of aging on IESC and identify new mechanisms, biomarkers, and potential therapeutic targets to better monitor and promote successful IESC and intestinal aging.