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，对损伤后上皮修复和再生至关重要。IESC和肠上皮更新或修复能力的衰老相关改变可能导致感染，损害消化和吸收能力，影响对结直肠癌的易感性，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，对年龄诱导的IESC变化的理解受到阻碍。该计划将使用Sox9-EGFP报告小鼠，其中Sox9-EGFP表达水平不同，可以直接可视化和分离IESC、祖细胞、肠内分泌细胞（EEC）和其他分化的肠上皮细胞。通过使用Sox9-EGFP小鼠，我们获得了与衰老相关的IESC扩张的直接证据，以及胰岛素样生长因子1 （IGF1）有效且特异性地促进年轻成人损伤后IESC再生的证据。IGF1、IGF2或胰岛素水平深刻影响寿命，并可能决定多能干细胞或组织限制性干细胞的存活和数量，用于组织修复和完整性。igf和胰岛素通过IGF1受体（IGF1R）或胰岛素受体（IR）进行信号传递。IR以IR- b亚型和IR- a亚型的形式存在，IR- b亚型介导胰岛素的代谢作用，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和其他分化肠上皮谱系中的不同作用。将与Sox9-EGFP小鼠杂交，在肠上皮中具有完全绒毛蛋白（VC）介导的缺失IR （VC-IR¿/¿）或IGF1R （VC-IGF1R¿/¿）的小鼠，用于验证一个中心假设，即IR通过限制IGF1R的有丝分裂抗凋亡作用和促进维持分化功能来保护IESC和肠上皮免受衰老诱导的功能障碍。Aim# 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，以及这些衰老细胞在损伤后再生的能力；Aim# 2将定义IR缺失对IESC、祖细胞、EEC、分化谱系的影响，以及在正常衰老过程中或损伤后IESC和上皮再生期间对胰岛素或igf的反应；Aim#3将定义IGF1R缺失对IESC和祖细胞、EEC的影响；分化谱系和对胰岛素或igf在正常衰老过程中的反应，或在IESC和损伤后上皮再生过程中。这些发现将从根本上推进我们对衰老对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IESC和肠道衰老的成功。

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.