Characterization of the dedifferentiation process induced by newt regeneration derived extracts

翡翠纽螈，具有再生结构的能力(例如。四肢、尾巴、脊髓、晶状体)，因受伤或截肢而丧失。损伤部位的细胞恢复为胚胎型细胞，然后可以增殖并改变命运，重建丢失的结构。这个过程被称为去分化，是再生过程的关键之一，了解去分化背后的分子基础可能有助于增强人类的再生潜力。研究表明，当用纽特再生提取物处理哺乳动物肌肉细胞时，它们重新进入细胞周期，并从多核结构中解体，产生增殖的单核细胞。然而，这个过程并不是很有效，许多细胞在这个过程中经历了细胞死亡。目前这项研究提案的目的是研究纽特细胞的去分化过程，以更好地了解如何改善人类的去分化和再生。我们将研究培养中的Newt A1肌肉细胞，这些细胞通过向培养液中添加血清或用Newt提取液处理而被诱导去分化。我们将使用一种称为代表性差异分析(RDA)的技术来分离和表征在Newt去分化过程中具有潜在调节作用的基因。我们提出了一种全面的策略，用于分离、优先排序和分析在去分化过程中具有调节作用的候选基因。我们将1)对细胞系进行RDA以确定启动去分化的候选基因，2)优先选择cDNA以确定相关候选基因，3)对候选基因在再生中进行功能分析。对去分化机制的了解将在退行性疾病和神经元损伤中有巨大的治疗应用。

The newt, Notophthalmus viridescens, has the ability to regenerate structures (eg. limbs, tail, spinal cord, lens) which are lost to injury or amputation. Cells at the site of the injury revert back to an embryonic type of cell that can then proliferate and switch fate to rebuild the lost structure. This process, called dedifferentiation, is one of the keys to the regeneration process, and understanding the molecular basis behind dedifferentiation could help to enhance regenerative potential in humans. Studies have shown that mammalian muscle cells, when treated with newt regeneration extracts, re-enter the cell cycle and break-up from their multinucleated structures to yield proliferating mononuclear cells. This process is not very efficient, however, and many of the cells undergo cell death in the process. The aim of the current research proposal is to study the dedifferentiation process in newt cells in order to better understand how to improve dedifferentiation and regeneration in humans. We will study newt A1 muscle cells in culture that have been induced to dedifferentiate by adding serum to the medium or by treatment with newt-derived extracts. We will use a technique called Representational Difference Analysis (RDA) to isolate and characterize genes with potential regulatory roles during the newt dedifferentiation process. We present a comprehensive strategy for the isolation, prioritization and analysis of candidate genes with regulatory roles in the dedifferentiation process. We will 1) Conduct RDA on cell lines to identify dedifferentiation-initiating candidate genes, 2) Prioritize cDNAs to identify relevant candidates and 3) Perform functional analysis of candidate genes in regeneration. An understanding of the mechanisms involved in dedifferentiation will have tremendous therapeutic applications in degenerative disease and in neuronal injury.