Assessing lineage decisions of musculoskeletal progenitor cells with aging

描述（由申请人提供）： 肌肉骨骼系统随年龄的恶化导致肌肉减少症和骨质疏松症，这是一个多因素的现实，对虚弱的老年人的肌肉力量、活动性和功能独立性的丧失具有显著的临床限制。小鼠模型可以提供一个窗口的环境和遗传因素，有助于该过程。这项资助将测试两个独立但趋同的假设：一个模型基于谱系调控，重点是一个共同的祖细胞与发展成肌，成骨或脂肪谱系的能力，第二个模型中，入侵的脂肪细胞对成熟肌肉组织的免疫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其主要或默认方向（骨骼和肌肉）到次要替代方向（脂肪）的谱系进展中断。为了测试该模型，将标记细胞分化的特定水平（Col3.6，骨; AP 2，脂肪; myoD，肌肉;平滑肌肌动蛋白，SMA，肌成纤维细胞）的GFP-报告基因构建体引入C57 BI/6（受影响的）和129 P3/J（抗性）小鼠中，并将动物年龄延长至10和24个月。通过将报道构建体与互补色组合，将在原代细胞培养模型中评估谱系从祖细胞发展为完全成熟的骨或肌肉细胞的能力，或祖细胞的分支点选择原代或脂肪细胞谱系的能力。将使用相同培养孔的重复荧光成像在真实的时间内评估分化的克里思和程度，并且将通过FAC分析获得处于限定发育水平的细胞的比例。体外分析将提示对遗传和年龄相关因素特别敏感的分化水平，并且将通过FAC分离的细胞群的微阵列分析来检查该效应的潜在遗传介质。将通过对年轻和老年宿主的祖细胞移植实验以及评估内源性祖细胞谱系选择的BrdU标记研究对培养结果进行体内验证。为了测试脂肪细胞原位组织破坏的第二个模型，将对两个实验组的骨和肌肉中GFP阳性脂肪细胞的出现以及肌纤维细胞内脂肪细胞与无序微结构的空间相关性进行表型分析。在这项资助中开发的工具，技术和分子概念将为未来的小鼠QTL研究提供基础，旨在绘制有助于骨骼健康年龄相关变化的遗传因素。

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.