Intestinal Stem Cell Survival and Renewal Coordinately Regulated by PUMA and p21

描述(申请人提供)：成体组织干细胞被认为在大多数组织损伤后负责维持组织动态平衡和再生。小肠是对放射性损伤最敏感的器官之一，是腹部和盆腔放射治疗的主要并发症，目前尚无有效的治疗方法。肠道干细胞(ISCs)位于隐窝的底部或接近底部，位于一个独特但不明确的微环境(“生态位”)中，该环境由来自骨髓的几种细胞类型组成。然而，直到最近，他们的身份仍然难以捉摸。遗传学证据表明，分布于Lgr5标记的Paneth细胞和Bmi-1标记的Paneth细胞上方的隐窝柱状细胞(CBC)可能代表ISCs的不同亚群。因此，它们的功能如何通过内源性和外源性(生态位相关)程序参与损伤后的肠道再生尚不清楚。我们和其他人的工作表明，仅BH3蛋白PUMA和细胞周期蛋白依赖的激酶抑制物p21调节肠道和造血系统的存活和再生，骨髓调节肠道放射敏感性。我们假设，细胞凋亡和细胞周期停滞都通过内在和外在机制对辐射后肠道干细胞的存活和再生起关键调控作用。在这项建议中，我们将结合一种新的ISC谱系标记和追踪小鼠模型和各种基因敲除和移植模型来1)确定ISCs在辐射后隐窝再生中的作用，并开发其分离和鉴定方法；2)展示PUMA和p21在调节ISCs存活和再生方面的潜在协同作用；3)确定骨髓对肠道再生的潜在贡献。我们相信，我们的研究可以更好地了解ISC生物学，并为分离、表征和操纵这些关键细胞用于治疗目的提供新的模型和策略。 与公共卫生相关：拟议的研究旨在确定辐射后肠道干细胞损伤、存活和再生的潜在分子机制，并开发分离和表征它们的方法。这样的研究可以帮助制定应对放射治疗或意外辐射暴露造成的肠道损伤的策略。

DESCRIPTION (provided by applicant): Adult tissue stem cells are believed to be responsible for maintaining tissue homeostasis and regeneration following injury in most tissues. The small intestine is one of the most sensitive organs to radiation-induced damage, which is the major complication in abdominal and pelvic radiotherapy with no effective treatment. The intestinal stem cells (ISCs) are located at or near the bottom of crypts in a unique yet poorly defined microenvironment ("niche") composed of several cell types derived from the bone marrow (BM). However, their identity remained elusive until recently. Genetic evidence demonstrated that the crypt-based columnar cells (CBCs) interspersed among Paneth cells marked by Lgr5 and some +4 cells immediately above Paneth cells marked by Bmi-1, represent perhaps distinct subsets of ISCs. Therefore, how their functions are regulated by intrinsic and extrinsic (niche-related) programs to participate in intestinal regeneration following injury is poorly understood. Work from us and others indicate that the BH3-only protein PUMA and cyclin-dependent kinase inhibitor p21 regulate the survival and regeneration of intestinal and hematopoietic systems, and the bone marrow modulates intestinal radiosensitivity. We hypothesize that both apoptosis and cell cycle arrest critically regulate the survival and regeneration of intestinal stem cells following radiation through intrinsic and extrinsic mechanisms. In this proposal, we will combine a novel ISC lineage marking and tracing mouse model with various knockout and transplantation models to 1) define the role of ISCs in crypt regeneration following radiation and develop assays for their isolation and characterization; 2) demonstrate a potential coordination of PUMA and p21 in modulating the survival and regeneration of ISCs; and 3) define the potential bone marrow contributions to intestinal regeneration. We believe that our studies can lead to a better understanding of ISC biology, and new models and strategies for isolation, characterization and manipulation of these critical cells for therapeutic purposes. PUBLIC HEALTH RELEVANCE: The proposed studies aim to determine the underlying molecular mechanisms governing the injury, survival and regeneration of intestinal stem cells following radiation, and to develop assays for their isolation and characterization. Such studies can help devise strategies to combat intestinal damage caused by radiotherapy or accidental radiation exposure.