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（由适用提供）：肢体损失是美国的主要健康问题，近200万患者生活在主要肢体截肢的后果。预计此数字将随关键危险因素的增加而增加，并且没有设计生物疗法来解决此问题。尽管人类在四肢和其他关键结构中的再生能力极为有限，但Axolotl Salamanders可以在整个生命中再生整个四肢。 Axolotl肢体在解剖学上与人类四肢相似，它们通过相似的机制发展。了解能够使Axolotl肢体再生能够对分子机制有透彻的了解，可以对可能在再生医学中采取的未来方法提供关键的见解，这反过来又可能彻底改变了面临截肢患者提供的治疗选择。这种彻底的机械理解已经逃避了迄今为止的研究人员，因为缺乏用于实验操纵基因表达的工具。但是，在过去的八年中，我们和其他人开发了强大的分子遗传工具，这些工具在Axolotls中在体内运行。我们建议利用这些发展来重新考虑脊椎动物肢体再生的长期和重要问题。在Axolotls中，最早的事件之一是在整个树桩上形成专门的伤口表皮。在此伤口表皮下，内部组织的祖细胞池形成。囊泡瘤对于肢体再生至关重要，但是它们的创造和生长知之甚少，并且很有可能这两个方面在伤口表皮的控制之下。伤口表皮及其分子因素的精确作用是难以捉摸的，因为缺乏研究这些问题的工具。通过大规模的RNA测序努力扩展到单细胞水平，我们已经确定了候选基因，其表达在伤口表皮中高度富集在表皮与Blastemas细胞中，并且在所有其他时间进行了采样。在这里，我们建议利用这些数据集以及我们最近开发的逆转录病毒系统，用于感染体内的axolotls 回答有关伤口表皮作用并检查五个特定基因的特定问题。在特定的目标1中，我们将确定使用逆转录病毒以标记活化细胞的逆转录病毒后的肌肉祖细胞激活软骨和肌肉祖细胞是否需要伤口表皮。在特定的目标2中，我们将测试五个富含表皮的富含基因的功能，以引起没有伤口表皮的四肢的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，从而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鉴定伤口表皮的关键分子成分的可行性。进行这项研究实现了未来的实验，旨在对支持伤口表皮功能的分子途径进行更密集的解剖，IT并将为考虑这些过程在哺乳动物背景下的作用奠定基础。