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

描述(申请人提供)：虽然适当的机械负荷，如运动，有利于维持肌腱的动态平衡，但慢性、重复的机械负荷可能会导致肌腱病的发展，这是慢性残疾的主要原因，仅在美国就有数百万人受到影响。肌腱病的典型组织病理学特征已被确定，包括脂肪细胞堆积、粘液变性和组织钙化，无论是单独的还是合并的。这些发现提示存在不同于表达成纤维细胞表型的肌腱细胞的多种表型的细胞。在我们母项目的研究过程中，我们已经鉴定了肌腱干细胞，它构成了肌腱细胞的一个亚群，可以进行成脂、成软骨和成骨分化。因此，我们现在有条件将干细胞生物学引入到肌腱病发病机制的研究中。此外，越来越多的证据表明，成体干细胞在组织病理学中的突出作用，如肿瘤发生。因此，在这项研究中，我们的目标是检验一种新的假设，即肌腱干细胞在肌腱疾病的发展中发挥重要作用，它通过在肌腱上施加慢性、重复的机械负荷而向“错误的”非腱细胞方向分化(例如，脂肪细胞、软骨细胞和骨细胞)。因此，本项目的具体目的是：通过跑步机运动来确定慢性、重复的机械负荷对小鼠肌腱干细胞分化的影响。我们将利用组织化学和免疫组织化学方法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，鉴定肌腱干细胞的分化状态，并通过基因和蛋白表达分析(RT-PCR和Western印迹)、流式细胞仪(FACS)分析和免疫细胞化学检测分化的肌腱干细胞(腱细胞和非腱细胞)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的分化状态，并验证生化分析的结果。这项研究是我们母项目的合乎逻辑的扩展，也是第一次调查肌腱干细胞在肌腱病发展中的作用。这项研究的发现将为肌腱病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开发，肌腱变性是肌腱病后期的标志。

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.