Molecular control of intestinal regeneration

描述（由申请人提供）：人类和大多数脊椎动物再生丢失或受损器官的能力受到严重限制。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，一组与脊椎动物密切相关的动物。我们一直以光壳海参为模型系统来研究肠再生。这些动物可以再生其完整的消化道后，自动内脏的过程。我们的实验室已经表征了肠再生过程和相关的细胞事件。近年来，我们已经开始确定可能控制再生过程的不同方面的基因，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。这些实验导致了候选基因的鉴定差异表达在肠再生。目前的研究集中在其中的20个基因上。我们建议使用这些候选基因来研究肠再生的分子控制。我们打算验证他们的差异表达和表征他们的序列，空间和时间的表达模式，使用计算机方法以及QRT-PCR，Western印迹，原位杂交和免疫组织化学。这将导致确定它们表达的细胞或组织成分以及基因的推定功能。更重要的是，我们将通过使用干扰RNA（RNAi）结合各种标记物的敲低实验来确定基因的功能，这些标记物用于确定再生过程中发生的细胞过程，例如：组织细胞成分和形态的变化，再生芽基、肠腔、肠神经系统和组织神经支配的形成，以及细胞群、迁移、分裂死亡、去分化和生长等等。我们的研究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。我们期望我们的实验将提供可用于研究脊椎动物再生能力的局限性的信息，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.