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

描述（由申请人提供）：本研究旨在确定细胞外基质（ECM）在骨骼肌老化中的作用。衰老过程中发生的骨骼肌质量和强度的丧失是不断增长的老年人口面临的一个主要健康问题。骨骼肌稳态是组织的常驻干/祖细胞的责任，称为卫星细胞或肌肉祖细胞（MPC）。随着年龄的增长，MAPCs的再生能力减弱。在确定MPC再生能力如何丧失时，必须考虑细胞内在和细胞外在变化都可能影响MPC。细胞的外在因素包括环境成分，如可溶性因子，邻近细胞和ECM，每一种都可以随着年龄的增长而经历许多变化。这里描述的研究调查ECM年龄对MPC进行骨骼肌组织再生维持所需过程的能力的影响。我们推测骨骼肌细胞外基质在衰老过程中的改变导致了MPC用于维持骨骼肌稳态的再生机制的失调。这是第一个研究，以隔离ECM的影响，从细胞和可溶性成分对肌肉再生的作用，作为年龄的函数。在这项拟议的研究中，我们将利用ECM和MPC从不同年龄的动物，研究它们之间的相互作用和ECM对祖细胞功能的影响，以及其对功能组织形成的影响。选择大鼠作为模型生物体，以最大限度地减少遗传变异性，并获得对ECM和MPC年龄的精确控制。使用我们实验室开发的方法，将年轻成年大鼠（<12个月）和老年大鼠（>24个月）的骨骼肌组织脱细胞，这是一个去除细胞物质同时保持ECM完整的过程。ECM年龄对年轻和老年MPC的生长行为和再生能力的影响将使用两种体外模型进行研究。在第一种方法中，将从脱细胞组织中提取ECM，并用于涂覆组织培养皿，将MPC接种到组织培养皿上。在第二种方法中，脱细胞组织切片将用作支架，MPC将被接种到支架上，三维（3D）组织将在生物反应器系统中的生理负载条件下生长。在两种培养系统中，将老年或年轻大鼠MPC以2 × 2析因实验设计接种到老年或年轻ECM的板或支架上。在这两个系统中，将测量ECM年龄对MPC的肌生成、增殖和分化的影响，包括监测已知肌源性分化所需的蛋白质的表达。此外，将测定整体基因表达，以确定MPC的基因表达如何根据培养系统ECM组分的年龄发生变化。最后，将测量MPC-支架构建体的功能以确定功能性工程化组织的形成如何受到ECM组分的老化的影响。完成后，这项研究将提供重要的洞察ECM年龄在调节对老年人健康至关重要的干/祖细胞群体的再生能力方面所起的作用。这些数据将为将来研究细胞微环境中的成分差异提供基础，这些成分差异对再生肌细胞具有刺激和/或抑制作用。公共卫生相关性：骨骼肌质量和力量的老化过程的特点的损失代表了一个重要的健康问题，面临着不断增长的老年人口。一个基本的问题，仍然没有答案是年龄相关的变化，肌肉干细胞的物理微环境，或细胞外基质，在骨骼肌老化中发挥的作用。这项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，并确定其表达和活性受到细胞微环境年龄相关变化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.