EPIDERMAL FACTORS THAT PROMOTE INTERNAL TISSUE PROGENITOR ACTIVATION FOLLOWING AMPUTATION

截肢后促进内组织祖细胞激活的表皮因素

• 批准号: 9253350
• 负责人: JESSICA L. WHITED
• 金额: $ 8.85万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-05-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9253350
• 关键词: Address; Ambystoma; Amputation; Anatomy; Animals; Biological Response Modifier Therapy; Candidate Disease Gene; Cartilage; Cell Cycle; Cells; Data Set; Development; Differentiation Antigens; Dissection; Epidermis; Event; Future; Gene Expression; Genes; Growth; Health; Human; Injury; Label; Lead; Limb Development; Limb structure; Mammals; Methodology; Mitotic; Molecular; Molecular Genetics; Muscle; Natural regeneration; Organism; Pathway interactions; Patients; Process; Regenerative Medicine; Research; Research Personnel; Resources; Retroviridae; Risk Factors; Role; Salamander; Stem cells; Structure; Surgical sutures; System; Testing; Thick; Tissue Sample; Tissues; Wound Healing; base; blastema; cartilage cell; design; experimental study; in vivo; insight; interest; limb amputation; limb regeneration; molecular marker; novel; progenitor; programs; public health relevance; regenerative; satellite cell; tool; transcriptome; transcriptome sequencing; wound epidermis

 DESCRIPTION (provided by applicant): Limb loss is a major health concern in the U.S. with nearly two million patients living with the consequences of a major limb amputation. This number is expected to rise with increases in key risk factors, and no biological therapeutics has been devised to address this problem. While humans have exceedingly limited regenerative abilities in limbs and other key structures, axolotl salamanders can regenerate entire limbs throughout their lives. Axolotl limbs are anatomically similar to human limbs, and they develop by similar mechanisms. Gaining a thorough understanding of the molecular mechanisms that enable axolotl limb regeneration stands to offer critical insights into future approaches that may be taken in regenerative medicine, which could in turn revolutionize the treatment options offered to patients facing amputation. This thorough mechanistic understanding has evaded researchers to date because of a paucity of tools available for experimentally manipulating gene expression in axolotls. However, within the last eight years, we-and others-have developed powerful molecular genetic tools that are operational in vivo in axolotls. We propose to leverage these developments to take a fresh look at the longstanding and important question of vertebrate limb regeneration. In axolotls, one of the earliest events post-amputation is the formation of a specialized wound epidermis across the stump. Beneath this wound epidermis, progenitor cell pool for internal tissues, the blastema, forms. Blastemas are critical for limb regeneration, but their creation and growth are poorly understood, and there is a strong possibility that both aspects are under the control of the wound epidermis. Precise roles for wound epidermis and its molecular factors have been elusive because of a lack of tools for studying these questions to date. Through a massive RNA-sequencing effort extended to single-cell level, we have identified candidate genes whose expression is highly enriched in the wound epidermis versus blastema cells and all other tissues sampled. Here we propose to leverage this data set as well as our recently-developed retrovirus system for infecting axolotls in vivo to answer specific questions about the role of wound epidermis and to examine five specific genes. In Specific Aim 1, we will determine if wound epidermis is required for activation of cartilage and muscle progenitors post-amputation using a retrovirus to mark activated cells. In Specific Aim 2, we will test the sufficiency of five wound-epidermis-enriched genes to cause dedifferentiation or stem cell activation in limbs without wound epidermis. These experiments will allow us to establish a system whereby we can study critical cellular events downstream of the wound epidermis, and it test the feasibility of using this approach to identify key molecular components of wound epidermis. Performing this research enable future experiments aimed at more intensive dissection of the molecular pathways that support the wound epidermis functions, it and will lay the groundwork for considering the role of these processes in the mammalian context.

 描述（由申请人提供）：在美国，肢体丧失是一个主要的健康问题，近 200 万患者承受着严重截肢的后果。随着关键危险因素的增加，这一数字预计还会上升，而且目前还没有设计出生物疗法来解决这个问题。虽然人类的四肢和其他关键结构的再生能力极其有限，但蝾螈可以在一生中再生整个四肢。蝾螈的四肢在解剖学上与人类四肢相似，并且通过相似的机制发育。全面了解使蝾螈肢体再生的分子机制，将为再生医学中可能采用的未来方法提供重要的见解，这反过来又可能彻底改变为面临截肢的患者提供的治疗选择。迄今为止，由于缺乏可用于实验性操纵蝾螈基因表达的工具，研究人员无法对这种彻底的机制进行理解。然而，在过去的八年里，我们和其他人已经开发出了强大的分子遗传工具，可以在蝾螈体内发挥作用。我们建议利用这些进展重新审视脊椎动物肢体再生这一长期存在的重要问题。 在蝾螈中，截肢后最早的事件之一是在残肢上形成特殊的伤口表皮。在伤口表皮下方，形成了内部组织的祖细胞池，即芽基。胚细胞对于肢体再生至关重要，但人们对它们的产生和生长知之甚少，并且很可能这两个方面都受到伤口表皮的控制。由于迄今为止缺乏研究这些问题的工具，伤口表皮及其分子因素的精确作用一直难以捉摸。通过扩展到单细胞水平的大规模 RNA 测序工作，我们已经确定了候选基因，与芽基细胞和所有其他采样组织相比，其表达在伤口表皮中高度富集。在这里，我们建议利用该数据集以及我们最近开发的逆转录病毒系统来体内感染蝾螈 回答有关伤口表皮作用的具体问题并检查五个特定基因。在具体目标 1 中，我们将使用逆转录病毒标记激活的细胞，确定截肢后软骨和肌肉祖细胞的激活是否需要伤口表皮。在具体目标 2 中，我们将测试五个富含伤口表皮的基因是否足以在没有伤口表皮的四肢中引起去分化或干细胞激活。这些实验将使我们能够建立一个系统，通过该系统我们可以研究伤口表皮下游的关键细胞事件，并测试使用这种方法来识别伤口表皮关键分子成分的可行性。进行这项研究使得未来的实验能够更深入地剖析支持伤口表皮功能的分子途径，这将为考虑这些过程在哺乳动物环境中的作用奠定基础。