Defining the role of tendon stem cells in the development of tendinopathy

描述（由申请人提供）：虽然适当的机械负荷，如运动，有利于维持肌腱稳态，但慢性、重复的机械负荷可能导致肌腱病变的发展，这是导致慢性残疾的主要原因，仅在美国就有数百万人受到影响。肌腱病变的典型组织病理学特征已被确定，包括脂质细胞积聚、粘液变性和组织钙化，无论是单独的还是联合的。这些发现表明存在多种表型的细胞，不同于表达成纤维细胞表型的驻留细胞。在我们的母项目的调查过程中，我们已经确定了肌腱干细胞，它构成肌腱细胞的一个亚群，可以经历成脂肪、成软骨和成骨的分化。因此，我们现在可以很好地将干细胞生物学引入我们对肌腱病变发病机制的研究中。此外，越来越多的证据表明成体干细胞在组织病理（如肿瘤发生）中的重要作用。因此，在本研究中，我们的目标是验证一个新的假设，即肌腱干细胞在肌腱受到慢性、重复性机械负荷时，通过向“错误”的非肌腱细胞方向（如脂肪细胞、软骨细胞和骨细胞）分化，在肌腱病变的发展中发挥重要作用。因此，本项目的具体目的是：为了确定通过跑步机运行的慢性、重复性机械负荷对小鼠肌腱干细胞（MTSCs）分化的影响，我们将使用组织化学和免疫组织化学来检测肌腱切片中肌腱基质和细胞表型的变化。我们还将从肌腱样本中提取肌腱细胞，表征MTSCs的分化状态，并通过基因和蛋白质表达分析（RT-PCR和Western blot）、流式细胞术（即FACS）分析和免疫细胞化学确定分化的MTSCs（肌腱细胞和非肌腱细胞）的表型。最后，我们将使用一种新的生物物理方法，细胞牵引力显微镜（CTFM），来评估MTSC分化状态并验证生化分析结果。这项研究是我们母项目的逻辑延伸，也是第一个研究肌腱干细胞在肌腱病变发展中的作用的研究。这项研究的发现将为肌腱病变的确切发病机制带来新的见解，并可能导致减缓或逆转肌腱变性的新疗法的发展，肌腱变性是肌腱病变晚期的标志。

DESCRIPTION (provided by applicant): While appropriate mechanical loading, such as exercise, is beneficial for maintaining tendon homeostasis, chronic, repetitive mechanical loading may lead to the development of tendinopathy, which is the leading cause of chronic disabilities that affects millions of people just in the United States alone. The typical histopathological features of tendinopathy have been identified, including accumulation of lipid cells, mucoid degeneration, and tissue calcification, either alone or in combination. These findings suggest the presence of cells with multi-phenotypes that differ from residential tenocytes, which express the fibroblast phenotype. During the course of investigation for our parent project, we have identified tendon stem cells, which constitute a sub-population of tendon cells and can undergo adipogenic, chondrogenic, and osteogenic differentiation. Therefore, we are now in good position to introduce stem cell biology into our investigation of the pathogenic mechanisms for the development of tendinopathy. Furthermore, accumulating evidence has pointed to the prominent role of adult stem cells in tissue pathologies, such as tumorigenesis. Thus, in this study, we aim to test a novel hypothesis that tendon stem cells play a major role in the development of tendinopathy by differentiating in "wrong" non-tenocyte directions (e.g., adipocytes, chondrocytes, and osteocytes) in response to chronic, repetitive mechanical loading placed on tendons. Therefore, the specific aim of this project is: To determine the effect of chronic, repetitive mechanical loading via treadmill running on the differentiation of mouse tendon stem cells (MTSCs) We will use histochemistry and immunohistochemistry to detect changes in the tendon matrix and cell phenotype in tendon sections. We will also extract tendon cells from tendon samples, characterize the differentiation state of MTSCs, and determine phenotypes of differentiated MTSCs (tenocytes and non- tenocytes) by gene and protein expression analysis (RT-PCR and Western blot), flow cytometry (i.e. FACS) analysis, and immunocytochemistry. Finally, we will use a novel biophysical approach, cell traction force microscopy (CTFM), to assess MTSC differentiation state and verify the results of biochemical analysis. This study is a logical extension of our parent project and is the first to investigate the role of tendon stem cells in the development of tendinopathy. The findings of this study will bring new insights into the precise pathogenesis of tendinopathy and may lead to the development of novel therapies for slowing down or reversing tendon degeneration, a hallmark of tendinopathy at later stages.