Assessing lineage decisions of musculoskeletal progenitor cells with aging

描述(申请人提供)：肌肉骨骼系统随着年龄的增长而恶化，导致骨质疏松症和骨质疏松症，这是一个多因素的现实，对虚弱的老年人的肌肉力量、活动能力和功能独立性的丧失具有显著的临床限制。小鼠模型可能提供一个窗口，了解环境和遗传因素对这一过程的影响。这笔赠款将检验两个独立但趋同的假说：一个基于谱系调控的模型，专注于具有向成肌、成骨或成脂谱系发展的共同祖细胞，以及第二个模型，在该模型中，入侵的脂肪细胞对成熟肌肉组织的免疫学和收缩状态产生不利影响。第一个模型设想，与年龄相关的基因活性变化会导致从主要或默认方向(骨骼和肌肉)到次要替代方向(脂肪)的谱系进程中断。为了测试这一模型，GFP-Report构建了标记特定细胞分化水平(Col3.6，骨；AP2，脂肪；myoD，肌肉；平滑肌肉活动。SMA，肌成纤维细胞)的结构，并将其引入C57BI/6(受影响)和129只P3/J(耐药)小鼠，这些动物将分别出生到10和24个月。通过将报告构建体与互补颜色相结合，将在原代细胞培养模型中评估祖细胞从祖细胞发展到完全成熟的骨或肌肉细胞的能力，或者祖细胞的分支点选择初级或脂肪细胞谱系的能力。分化的速度和程度将使用相同培养井的重复荧光成像进行实时评估，并通过FAC分析获得处于特定发育水平的细胞的比例。体外分析将提出对遗传和年龄相关因素特别敏感的分化水平，并将通过对FAC分离细胞群体的微阵列分析来检验这种影响的潜在遗传中介因素。培养结果的体内验证将通过将祖细胞移植到年轻和老年宿主中的实验来进行，并通过BrdU标记研究来评估内源性祖细胞的谱系选择。为了测试脂肪细胞原位组织破坏的第二个模型，将对两个试验组进行GFP阳性脂肪细胞在骨骼和肌肉中的出现以及脂肪细胞与肌肉纤维细胞内紊乱微结构的空间相关性的表型。这笔赠款中开发的工具、技术和分子概念将为未来在小鼠身上进行QTL研究奠定基础，这些QTL研究旨在绘制有助于骨骼健康年龄相关变化的遗传因素图。

DESCRIPTION (provided by applicant): The deterioration of the musculoskeletal system with age that leads to sarcopenia and osteoporosis is a multi-factorial reality that has significant clinical limitations for muscle strength, mobility and loss of functional independence in frail older adults. Murine models may provide a window into the environmental and genetic factors that contribute to the process. This grant will test two independent yet converging hypothesis: one model based on lineage regulation that focuses on a common progenitor cell with the ability to develop into the myogenic, osteogenic or adipogenic lineages, and a second model in which invading adipocytes adversely influence the immunological and contractile status of mature muscle tissue. The first model envisions that age-related changes in gene activity result in a disruption of lineage progression from its primary or default direction (bone and muscle) to a secondary alternative direction (fat). To test this model, GFP-reporter constructs that mark specific levels of cellular differentiation (Col3.6, bone; AP2, fat; myoD, muscle; smooth muscle actin.SMA, myofibroblast) will be introduced in to C57BI/6 (affected) and 129 P3/J (resistant) mice and the animals will be aged to 10 and 24 months. By combining reporter constructs with complementary colors, the ability of the lineage to progress from a progenitor to fully mature bone or muscle cells, or the branch point of the progenitor to select the primary or adipocytic lineage will be assessed in a primary cell culture model. The tempo and extent of differentiation will be assess in real time using repetitive fluorescence imaging of the same culture well, and the proportion of cells at a defined levels of development will be obtained by FAC analysis. In vitro analysis will suggest levels of differentiation that are particularly sensitive to genetic and age-related factors and the potential genetic mediators of the effect will be examined by microarray analysis of the FAC isolated cell populations. In vivo validation of the culture results will be performed by progenitor transplantation experiments into young and aged hosts, and by a BrdU labeling study that assesses lineage choices of the endogenous progenitor cells. To test the second model of in-situ tissue disruption by adipocytes, the two experimental groups will be phenotyped for appearance of GFP positive fat cells in bone and muscle and for a spatial correlation of adipocytes with disorganized microstructure within muscle fiber cells. The tools, techniques and molecular concepts that are developed in this grant will provide a foundation for future QTL studies in mice designed to map genetic factors that contribute to age-related changes in skeletal health.