Cell-cycle commitment in muscle regeneration

肌肉再生中的细胞周期承诺

• 批准号: 10064946
• 负责人: Mingyu Chung
• 金额: $ 6.49万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-16 至 2023-07-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10064946
• 关键词: Address; Aging; Automobile Driving; Biochemical; Biological Assay; Biology; CDK4 gene; Cell Cycle; Cell Cycle Completion; Cell Cycle Progression; Cell Proliferation; Cell Separation; Cell physiology; Cells; Cyclin D1; Cyclin-Dependent Kinases; Cyclins; Data; Defect; Degenerative Disorder; Dependence; Epithelial Cells; Excision; Exhibits; Fellowship; Fibroblasts; Fluorescent in Situ Hybridization; G0 Phase; G1 Phase; Gene Expression; Generations; Genes; Genetic Transcription; Goals; Growth Factor; Harvest; Histology; Image; Image Analysis; Immunofluorescence Immunologic; In Vitro; Injections; Injury; Knockout Mice; Knowledge; Lead; Maintenance; Malignant Neoplasms; Maps; Mediating; Methodology; Methods; Mitogens; Modeling; Molecular; Muscle; Muscle satellite cell; Pathology; Process; RNA; Research; Role; Science; Scientist; Signal Transduction; Skeletal Muscle; Small Interfering RNA; System; Technology; Testing; Therapeutic; Time; Tissues; Western Blotting; Work; adult stem cell; base; career; conditional knockout; experimental study; extracellular; in vivo; in vivo evaluation; insight; mRNA Expression; muscle regeneration; protein expression; regenerative; response; satellite cell; skills; stem cell proliferation; stem cells; therapeutic target; time use; tissue regeneration; transcriptome; transcriptomics

PROJECT SUMMARY/ABSTRACT Mammalian tissues exhibit remarkable regenerative activity following injury, and defects in this process are associated with aging and degenerative diseases. Skeletal muscle regeneration requires a sustained proliferative response from muscle stem cells called satellite cells (SCs), yet it is unknown how this is achieved. Cellular proliferation is controlled by extracellular growth factors, or mitogens. The process of cellular proliferation, referred to as the cell cycle, involves cells exiting a non-proliferative G0 state and initiating the cell cycle in G1. At some point in G1, cells effectively escape the requirement for mitogens and commit to mitogen- independent completion of the cell cycle, but subsequent rounds of proliferation each require mitogen signaling during or just before G1. Insights into cell-cycle commitment mechanisms are primarily derived from in vitro studies of fibroblasts and immortalized epithelial cells, but it is unclear how relevant such systems are to the process of SC activation, where SCs exhibit a prolonged first G0/G1 phase, followed by multiple rounds of proliferation. Our preliminary work indicates that a mechanism of bistability, where a process remains indefinitely engaged despite the removal of mitogens, underlies cell-cycle commitment in the G0/G1 phase of SCs. The central hypothesis of this proposal is that a bistable G0/G1 mechanism underlies multiple rounds of mitogen-independent SC proliferation and effective muscle regeneration. The proposed work addresses the following specific aims: 1. Identify the mechanism of SC cell-cycle commitment through prolonged and multiple G0/G1 phases. 2. Establish mitogen signaling dynamics during muscle regeneration. 3. Determine the role of SC G0/G1 cell-cycle commitment during muscle regeneration. The mechanism of G0/G1 cell-cycle commitment will be identified using FACS-based SC isolation and ex vivo assays, including time-lapse imaging, immunofluorescence, and automated cell tracking and image analysis. In vivo mitogen signaling dynamics will be investigated using a muscle injury model and harvesting muscles for biochemical and transcriptomic analysis of growth factor availability and SC mitogen signaling. The functional role of SC G0/G1 cell-cycle commitment mechanisms in muscle regeneration will be tested using SC-specific conditional knockout mice and rescue by ectopic introduction of mitogens. Stanford is a leader in stem cell and regenerative sciences and will provide me with the technologies and collaborative network to develop into an independent research scientist. This research will uncover the fundamental relationship between mitogens and stem cell proliferation during muscle regeneration and may lead to therapeutic strategies for treating regenerative defects in aging and degenerative diseases.

项目总结/摘要 哺乳动物组织在损伤后表现出显着的再生活性，并且该过程中的缺陷是可接受的。 与衰老和退行性疾病有关。骨骼肌的再生需要持续的增殖 这是一种来自称为卫星细胞（SC）的肌肉干细胞的反应，但尚不清楚这是如何实现的。 细胞增殖受细胞外生长因子或有丝分裂原控制。细胞的过程 增殖，称为细胞周期，涉及细胞退出非增殖性G 0状态并启动细胞周期， 循环G1。在G1期的某个时间点，细胞有效地逃避了对有丝分裂原的需求，并致力于有丝分裂原。 细胞周期的独立完成，但随后的增殖每轮都需要有丝分裂原信号 在G1期间或之前。对细胞周期定型机制的了解主要来自体外 成纤维细胞和永生化上皮细胞的研究，但目前还不清楚这些系统是如何相关的， SC活化过程，其中SC表现出延长的第一个G 0/G1期，随后是多轮活化。 增殖我们的初步工作表明，一个机制的双稳态，其中一个过程仍然存在 尽管有丝分裂原被去除，但无限期地参与，是G 0/G1期细胞周期定型的基础。 SC.该提议的中心假设是，多轮谈判的基础是一个G 0/G1机制 不依赖于丝裂原的SC增殖和有效的肌肉再生。拟议工作涉及 具体目标如下： 1.通过延长和多个G 0/G1期确定SC细胞周期定型的机制。 2.在肌肉再生过程中建立有丝分裂原信号动力学。 3.确定肌肉再生过程中SC G 0/G1细胞周期定型的作用。 G 0/G1细胞周期定型的机制将使用基于FACS的SC分离和离体培养来鉴定。 分析，包括延时成像，免疫荧光，和自动化细胞跟踪和图像分析。在 体内有丝分裂原信号传导动力学将使用肌肉损伤模型进行研究， 生长因子可用性和SC促分裂原信号传导的生物化学和转录组学分析。功能 SC G 0/G1细胞周期承诺机制在肌肉再生中的作用将使用SC特异性 条件性基因敲除小鼠和通过异位导入有丝分裂原的拯救。 斯坦福大学是干细胞和再生科学的领导者，将为我提供技术， 合作网络，发展成为一个独立的研究科学家。这项研究将揭示 肌肉再生过程中有丝分裂原和干细胞增殖之间的基本关系， 从而导致用于治疗老化和退行性疾病中的再生缺陷的治疗策略。