Mechanisms of Skeletal Muscle Satellite Cell Regulation in Aging

描述（由申请人提供）：本提案的总体目标是确定衰老的全身环境无法招募肌肉特异性干细胞（称为卫星细胞（SC））导致骨骼肌肥大的机制。该提案的目的有两个：1）。将SC的固有特性与老化环境的影响分开，以及2.）测试GH、IGF-I、TNF-α和IL-6在环境年龄背景下对SC功能的作用。目标1的方法将采用交叉设计，年轻人原代SC培养物分别接受含有年轻人或老年人血清的培养基。同样，年老的SC将分别接受含有年轻或年老血清的培养基。为了考虑机械负载的影响，将使用FlexCell系统在循环拉伸和未拉伸条件下对细胞进行每次介质应用。这种方法的基本原理是，已经注意到SC功能随年龄的递减，但尚不清楚这些递减是由于SC本身还是环境的改变。此外，年龄的全身环境和机械负荷对SC表型的相互作用是未知的。将评价每个处理组的SC增殖（通过BrdU掺入）、凋亡（通过TUNEL染色）、分化（通过分化标志物的Western印迹）和融合（通过免疫组织化学技术），这反映了SC介导的肥大所需的四个过程。目的2的总体方法是操纵年轻和老年血清中GH、IGF-I、TNF-α和IL-6的水平，以确定各自对从老年受试者分离的体外SC的增殖、凋亡、分化和融合的影响。此外，将在拉伸和未拉伸条件下分析每个血清处理组。该方法的基本原理源于以下事实：先前已鉴定出几种候选分子在单独应用时会影响SC功能，但尚未确定这些特定因素在机械负荷下体外老化环境中的影响。总的来说，这些研究将提高我们对衰老中肌肉肥大的理解，并将指导未来的努力，以对抗与年龄相关的肌肉质量损失和脆弱。与公共卫生的相关性：这项工作将寻求确定老年人肌肉无法像年轻人肌肉那样生长和再生的机制。识别这种现象发生的机制将指导未来的努力，旨在治疗与年龄相关的肌肉质量损失。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to identify the mechanisms by which an aging systemic environment is unable to recruit muscle specific stem cells, termed satellite cells (SCs), to contribute to skeletal muscle hypertrophy. The Aims of this proposal are two-fold: 1.) to separate the intrinsic properties of the SC proper from the effects of the milieu in aging, and 2.) to test the role of GH, IGF-I, TNF-a, and IL-6 on SC function in the context of the age of the milieu. The approach of Aim 1 will utilize a crossover design, with young primary human SC cultures separately receiving media containing young or old human serum. Likewise, old SCs will separately receive media containing young or old serum. To account for the influence of mechanical load, each media application will occur with cells under both cyclically stretched and unstretched conditions using a FlexCell system. The rationale for this approach is that decrements in SC function with age have been noted, but it is unknown whether these decrements are due to the SC proper or an altered milieu. Further, the interaction of the age of the systemic environment and mechanical load on SC phenotype is unknown. Each treatment group will be evaluated for proliferation (via BrdU incorporation), apoptosis (via TUNEL staining), differentiation (via Western blotting of differentiation markers), and fusion (via immunohistochemical techniques) of SC, which are reflective of the four processes necessary for SC mediated hypertrophy. The overall approach of Aim 2 is to manipulate levels of GH, IGF-I, TNF-a, and IL-6 in both young and old serum to determine the influence of each over proliferation, apoptosis, differentiation, and fusion of in vitro SCs isolated from old subjects. In addition, each serum treatment group will be analyzed in the stretched and un-stretched conditions. The rationale for this approach stems from the fact that several candidate molecules have been previously identified that affect SC function when applied in isolation, however the effect of these specific factors in the context of an aged in vitro environment under mechanical load has not been determined. Overall, these studies will improve our understanding of muscle hypertrophy in aging and will direct future efforts to combat age-related loss of muscle mass and frailty. Relevance to Public Health: This work will seek to determine the mechanisms by which muscle from aged individuals is unable to grow and regenerate to the same extent as muscle from the young. Identification of the mechanisms by which this phenomenon occurs will direct future efforts aimed at therapy for age-related loss of muscle mass.