The role of ageing on the regulatory effects of skeletal muscle ECM...

描述(申请人提供)：这项研究试图确定细胞外基质(ECM)在骨骼肌衰老中所起的作用。在衰老过程中发生的骨骼肌质量和力量的丧失是日益增长的老年人口面临的一个主要健康问题。骨骼肌内稳态是组织的常驻干细胞/祖细胞，即卫星细胞或肌肉祖细胞(MPC)的责任。随着年龄的增长，MAPC的再生能力减弱。在确定MPC再生能力如何丧失时，必须考虑到细胞内和细胞外的变化都会影响MPC。细胞外的因素包括环境成分，如可溶性因子、邻近细胞和细胞外基质，每一种都可以随着年龄的增长而经历无数的变化。这里描述的研究调查了细胞外基质年龄对MPC执行骨骼肌组织再生维护所需过程的能力的影响。我们假设，衰老过程中骨骼肌细胞外基质的改变导致MPC用来维持骨骼肌内环境平衡的再生机制的错误调节。这是首次从细胞和可溶性成分中分离出细胞外基质对肌肉再生随年龄变化的影响的研究之一。在这项拟议的研究中，我们将利用不同年龄动物的ECM和MPC来研究它们之间的相互作用，以及ECM对祖细胞功能的影响，以及它对功能组织形成的影响。大鼠被选为模式生物，以最大限度地减少遗传变异，并获得对ECM和MPC年龄的精确控制。使用我们实验室开发的一种方法，年轻成年(12个月)和老年大鼠(24个月)的骨骼肌组织将被脱细胞，这一过程将去除细胞物质，同时保持细胞外基质完好无损。采用两种体外模型研究细胞外基质年龄对年轻和老年MPC生长行为和再生能力的影响。首先，从脱细胞组织中提取细胞外基质，用来覆盖组织培养皿，然后在上面种植基质细胞。在第二种方法中，脱细胞组织切片将被用作支架，在其上种植MPC，并在生物反应器系统中在生理负荷条件下生长三维组织。在这两种培养体系中，将老鼠或幼鼠的MPC按2×2析因实验设计种植到老鼠或幼鼠ECM的培养板或支架上。在这两个系统中，将测量ECM年龄对MPC的肌肉生成、增殖和分化的影响，包括监测已知的肌源性分化所需的蛋白质的表达。此外，还将分析全球基因表达，以确定MPC的基因表达如何根据培养系统中细胞外基质成分的年龄发生变化。最后，将测量MPC-支架结构的功能，以确定ECM组件的年龄如何影响功能性工程组织的形成。这项研究完成后，将对细胞外基质年龄在调节对老年人健康至关重要的干细胞/祖细胞群体的再生能力方面所起的作用提供重要的见解。这些数据将为未来研究细胞微环境中的成分差异提供基础，这些微环境为再生肌肉细胞提供刺激和/或抑制信号。公共卫生相关性：骨骼肌质量和力量的丧失是老龄化过程中的特征，是日益增长的老年人口面临的一个重要健康问题。一个尚未解答的基本问题是，肌肉干细胞的物理微环境或细胞外基质的年龄相关变化在骨骼肌衰老中所起的作用。本研究旨在确定衰老的细胞微环境如何影响骨骼肌祖细胞的再生能力，并确定与年龄相关的细胞微环境变化对其表达和活性产生不利影响的基因和蛋白质。

DESCRIPTION (provided by applicant): This research seeks to determine the role extracellular matrix (ECM) plays in the aging of skeletal muscle. The loss of skeletal muscle mass and strength that occurs during the aging process represents a major health issue facing a growing elderly population. Skeletal muscle homeostasis is the responsibility of the tissue's resident stem/progenitor cell known as the satellite cell or muscle progenitor cell (MPC). With age, the regenerative capacity of MAPCs is diminished. In determining how MPC regenerative capacity is lost, one must consider that both cell-intrinsic and cell-extrinsic changes can impact MPCs. Factors extrinsic to the cell include environmental components such as soluble factors, neighboring cells, and ECM, each of which can experience numerous changes with age. The research described here investigates the impact ECM age has on the ability of MPCs to carry out processes required for the regenerative maintenance of skeletal muscle tissue. We hypothesize that alteration of skeletal muscle ECM during the aging process results in misregulation of the regenerative mechanisms used by MPCs to maintain skeletal muscle homeostasis. This is among the first research to isolate the effects of ECM from cellular and soluble component effects on muscle regeneration as a function of age. In this proposed study, we will utilize ECM and MPCs from different ages of animals to study the interaction between them and the effect ECM has on progenitor cell function, as well as its impact on functional tissue formation. Rats have been chosen as the model organism to minimize genetic variability and gain precise control over the age of both the ECM and the MPCs. Using a method developed in our lab, skeletal muscle tissue from young adult (<12 months) and old rats (>24 months) will be decellularized, a process which removes cellular material while leaving ECM intact. The influence of ECM age on the growth behavior and regenerative capacity of young and old MPCs will be investigated using two in vitro models. In the first, ECM will be extracted from the decellularized tissue and used to coat tissue culture dishes onto which MPCs will be seeded. In the second, slices of decellularized tissue will be used as scaffolds onto which MPCs will be seeded and three-dimensional (3D) tissue will be grown under physiological loading conditions in a bioreactor system. In both culture systems, old or young rat MPCs will be seeded onto plates or scaffolds of old or young ECM in a two-by-two factorial experimental design. In both systems, the impact of ECM age on myogenesis, proliferation, and differentiation of the MPCs will be measured, including monitoring the expression of proteins known to be required for myogenic differentiation. Additionally, global gene expression will be assayed to determine how gene expression of MPCs is altered based on the age of the ECM component of the culture system. Finally, the function of MPC- scaffold constructs will be measured to determine how the formation of functional engineered tissue is affected by the age of the ECM component. When completed, this research will provide significant insight into the role ECM age plays in regulating the regenerative capacity of a stem/progenitor cell population vital to the health of elderly individuals. These data will provide the basis for future investigations of the compositional differences in the cell microenvironment that presents stimulatory and/or inhibitory cues to regenerative muscle cells. PUBLIC HEALTH RELEVANCE: The loss of skeletal muscle mass and strength characteristic of the aging process represents an important health problem facing a growing elderly population. A fundamental question that remains unanswered is the role that age-related changes to the physical microenvironment, or extracellular matrix, of muscle stem cells play in the aging of skeletal muscle. This research is designed to determine how an aging cellular microenvironment affects the regenerative capacity of skeletal muscle progenitor cells and to identify the genes and proteins whose expression and activity is adversely affected by age-related changes to the cell microenvironment.