Aging Intestinal Stem Cells and Insulin/IGF System

描述(申请人提供)：肠上皮干细胞(IESC)在一生中更新小肠和结肠上皮，对损伤后的上皮修复和再生至关重要。增龄相关的IESC的改变以及更新或修复肠上皮的能力可能易于感染，损害消化和吸收能力，影响对结直肠癌的易感性，并限制耐受化疗或放射治疗的能力。由于缺乏有效的IESC生物标志物，对IESC由年龄引起的变化的理解一直受到阻碍。这项建议将使用Sox9-EGFP报告鼠，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)和其他分化的肠道上皮细胞。使用Sox9-EGFP小鼠，我们有直接证据表明与衰老相关的IESC扩张，并且胰岛素样生长因子1(IGF1)有效且特异地促进年轻人损伤后的IESC再生。IGF1、IGF2或胰岛素的水平对寿命有深远的影响，并可能决定组织修复和完整性所需的多能或组织受限干细胞的存活和数量。IGF和胰岛素通过IGF1受体(IGF1R)或胰岛素受体(IR)传递信号。IR以IR-B亚型和IR-A亚型存在，IR-B亚型介导胰岛素的代谢效应，IR-A亚型可能与生长特别相关。初步数据显示，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细胞系中的不同作用。与Sox9-EGFP小鼠杂交，在肠上皮具有完全绒毛蛋白Cre(VC)介导的缺失型IR(VC-IR？/？)或IGF1R(VC-IGF1R？/？)的小鼠将被用来检验一项中心假设，即IR通过限制IGF1R的有丝分裂抗凋亡作用和促进维持的分化功能来保护IESC和肠上皮免受衰老诱导的功能障碍。目的#1将确定老化对IESC、祖细胞、EEC或分化的谱系的数量、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。目的#2将确定IR缺失对IESC和祖细胞、EEC、分化的谱系以及在整个正常衰老过程中或在IESC和损伤后的上皮再生过程中对胰岛素或IGFS的反应的影响。目的#3将定义IGF1R缺失对IESC和祖细胞、EEC、分化的谱系和对胰岛素或IGFS的反应在整个正常衰老过程中，或在IESC和损伤后的上皮再生过程中的影响。这些发现将从根本上提高我们对衰老对IESC的影响的了解，并确定新的机制、生物标记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.