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

描述（由申请人提供）：在大多数组织中，成体组织干细胞被认为负责维持组织稳态和损伤后的再生。小肠是对放射性损伤最敏感的器官之一，是腹盆腔放射治疗的主要并发症，目前尚无有效的治疗方法。肠干细胞（ISCs）位于隐窝底部或靠近隐窝底部，处于一个独特但定义不明确的微环境（“生态位”）中，该微环境由来自骨髓的几种细胞类型组成（BM）。然而，直到最近，他们的身份仍然难以捉摸。遗传证据表明，分布在Lgr5标记的Paneth细胞和紧邻Bmi-1标记的Paneth细胞上方的一些+4细胞之间的隐窝柱状细胞（CBCs）可能代表不同的ISCs亚群。因此，它们的功能是如何通过内在和外在（生态位相关）程序参与损伤后肠道再生的，目前尚不清楚。我们和其他人的研究表明，BH3-only蛋白PUMA和周期蛋白依赖性激酶抑制剂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阻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着关键的调节作用。在本研究中，我们将结合一种新的ISC谱系标记和追踪小鼠模型与各种敲除和移植模型，以1)确定ISC在放射后隐窝再生中的作用，并开发其分离和表征的检测方法；2)证明PUMA和p21在调节ISCs存活和再生中的潜在协同作用；3)明确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理解ISC生物学，并为这些关键细胞的分离，表征和操作提供新的模型和策略，用于治疗目的。

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.