Molecular control of intestinal regeneration

描述(申请人提供)：在人类和大多数脊椎动物中，再生丢失或受损器官的能力严重有限。相比之下，其他动物具有非凡的再生能力。其中包括棘皮动物，这是一组与脊椎动物密切相关的动物。我们一直以海参为模型系统来研究肠道再生。这些动物在经过自动剔除内脏的过程后，可以再生它们的完整消化道。我们的实验室已经确定了肠道再生过程和相关的细胞事件。近年来，我们已经开始通过使用分子工具，特别是EST数据库的构建和分析以及微阵列的使用，来鉴定可能控制再生过程的不同方面的基因。这些实验导致了在肠道再生过程中差异表达的候选基因的鉴定。目前的提案重点关注其中的20个基因。我们建议使用这些候选基因来研究肠道再生的分子调控。我们打算验证它们的差异表达，并利用计算机技术以及QRT-PCR、Western blotting、原位杂交和免疫组织化学等方法来表征它们的序列、空间和时间表达模式。这将导致确定它们在哪里表达的细胞或组织成分，以及基因的假定功能。更重要的是，我们将通过使用干扰RNA(RNAi)结合各种标记的敲除实验来确定基因的功能，这些标记用于确定再生过程中发生的细胞过程，如：组织细胞成分和形态的变化，再生胚泡、肠腔、肠道神经系统和组织神经的形成，以及细胞种群的变化，迁移、分裂、死亡、去分化和生长等。我们的结果将有助于发现与肠道再生相关的新基因，并表征它们在特定细胞和再生事件中的表达。此外，将确定它们的功能，并确定它们如何控制肠道再生过程的机制。我们预计，我们的实验将提供可用于研究脊椎动物再生能力限制的信息，并将作为开发调节再生过程的新疗法/药物的基础。

DESCRIPTION (provided by applicant): The capability to regenerate lost or damaged organs is severely limited in humans and most vertebrate animals. Other animals, in contrast, have remarkable regenerating abilities. Among these are the echinoderms, a group of animals closely related to vertebrates. We have been using the sea cucumber Holothuria glaberrima as a model system to study intestinal regeneration. These animals can regenerate their complete digestive tract following a process of auto-evisceration. Our laboratory has characterized the intestinal regeneration process and the associated cellular events. In recent years we have begun to identify genes that might control different aspects of the regeneration process by using molecular tools, particularly the construction and analysis of an EST database and the use of microarrays. These experiments have led to the identification of candidate genes differentially expressed during intestinal regeneration. The present proposal focuses on 20 of these genes. We propose to use these candidate genes to study the molecular control of intestinal regeneration. We intend to validate their differential expression and characterize their sequence, spatial and temporal expression patterns, using in silico methods as well as QRT-PCR, Western blotting, in situ hybridization and immunohistochemistry. This will lead to determining the cellular or tissue component where they are being expressed and a putative function for the genes. More importantly, we will determine the gene's function by knock-down experiments using interference RNA (RNAi) in conjunction with various markers that serve to determine the cellular processes that occur during regeneration such as: changes in tissue cellular components and morphology, formation of the regeneration blastema, intestinal lumen, enteric nervous system, and tissue innervation, as well as changes in cell populations, migration, division, death, dedifferentiation, and growth among others. Our results will help discover novel genes associated with intestinal regeneration and characterize their expression to particular cells and regenerative events. Moreover, their function will be determined, and mechanisms of how they might control the intestinal regeneration process will be identified. We expect that our experiments will make available information, that can be used to study the limitations of vertebrate regenerative capacities and that will serve as the basis for the development of new therapeutics/drugs that modulate regenerative processes.