Colocalization of gene expression and microscale tissue strains in live tendon explants using barcoded biosensors

项目摘要 肌腱病是一种慢性退行性疾病，因肌腱过度使用(即疲劳负荷)而发生。 除了机械损伤外，肌腱疲劳负荷还会诱导分解代谢酶的表达， 炎性细胞因子和异常基质成分(如软骨、脂肪和 钙沉积)，这会进一步削弱组织并推动退化进程。理解 为什么肌腱细胞不能恢复组织结构，反而加剧了退化，这对预防 肌腱病。普遍的假设是，过度使用肌腱的细胞反应适得其反 由局部组织损伤引起的改变的生物物理刺激的机械转导。然而，还有 对肌腱细胞在天然组织中机械转导的基本知识的缺乏 环境，因此，改变的生物物理刺激是否负责肌腱退化。至 为了解决这一知识鸿沟，本提案的目标是开发一种能够 局部机械刺激和细胞基因表达的同时时空测量 肌腱疲劳载荷。通过将腱细胞的反应与自然存在的机械刺激共同定位 细胞微环境，我们可以直接测试机械转导是否对负性负责 肌腱变性时观察到的细胞反应。实现这一目标的关键技术障碍 目标是开发一种能够动态检测细胞基因空间分布的生物传感器 在肌腱外植体中的表达。内源性荧光记者受到生产时间和成本的限制 一种针对特定目标基因的转基因小鼠。替代生物技术需要细胞转基因，这是 在细胞外基质密集的组织中效率低下。因此，我们将调查金纳米棒， 它们通过内吞作用内化，可以递送荧光标记的寡核苷酸探针来检测细胞 基因在肌腱外植体中的表达。在目标1中，我们将优化金纳米棒锁定核酸的设计 生物传感器及其在测量活体机械敏感基因中的灵敏度和空间准确性 肌腱外植体。此外，我们将使用FRET对探头和光谱建立条形码策略 混合算法，可同时测量多达10个目标基因。在目标2中，我们将使用这项技术来 确定对肌腱疲劳的退行性细胞反应是否在空间上与 局部机械刺激(即应变)。这个项目是创新的，因为我们的结果将确定刺激 调节对肌腱过度使用的负面细胞反应并帮助开发预防的新疗法 退化。此外，我们还将建立测量基因时空分布的能力 在具有致密细胞外基质(例如肌腱)的活体组织外植体中表达。这项技术将被搁置- 由于外植体模型通常用于研究肌肉骨骼，因此肌肉骨骼研究的突破口 由于致密的细胞外基质，需要细胞转染的组织和现有技术并不有效。

Project Summary Tendinopathy is a chronic degenerative disease that occurs in response to tendon overuse (i.e., fatigue loading). In addition to mechanical damage, tendon fatigue loading induces the expression of catabolic proteases, inflammatory cytokines, and the accumulation of abnormal matrix components (e.g., cartilaginous, fat, and calcium deposits), which further weaken the tissue and drive the progression of degeneration. Understanding why tendon cells fail to restore the tissue structure and instead exacerbate degeneration is critical for preventing tendinopathy. The prevailing hypothesis is that the counterproductive cellular response to tendon overuse results from mechanotransduction of altered biophysical stimuli resulting from local tissue damage. However, there is a fundamental lack of knowledge regarding tenocyte mechanotransduction in the native tissue environment and, therefore, whether altered biophysical stimuli are responsible for tendon degeneration. To address this knowledge gap, the objective of this proposal is to develop a live tissue explant model enabling simultaneous spatiotemporal measurement of local mechanical stimuli and cellular gene expression during tendon fatigue loading. By colocalizing the tenocyte response with the mechanical stimuli present in the native cellular microenvironment, we can directly test whether mechanotransduction is responsible for the negative cellular response observed with tendon degeneration. The critical technological obstacle to achieving this goal is the development of a biosensor that can dynamically detect the spatial distribution of cellular gene expression in tendon explants. Endogenous fluorescent reporters are limited by the time and cost of generating a transgenic mouse for specific genes of interest. Alternative biotechnologies require cell transfection, which is inefficient in tissues with a dense extracellular matrix. Therefore, we will investigate whether gold nanorods, which are internalized via endocytosis, can deliver fluorescently labeled oligonucleotide probes to detect cellular gene expression in tendon explants. In Aim 1, we will optimize the design of gold nanorod-locked nucleic acid biosensors and validate their sensitivity and spatial accuracy in measuring mechanosensitive genes in live tendon explants. Additionally, we will establish a barcode strategy with FRET-pair probes and a spectral unmixing algorithm to simultaneously measure up to ten target genes. In Aim 2, we will use this technology to determine whether the degenerative cellular response to tendon fatigue is spatially associated with changes in local mechanical stimuli (i.e., strains). This project is innovative because our results will identify the stimuli mediating the negative cellular response to tendon overuse and help develop novel therapies for preventing degeneration. Additionally, we will establish the ability to measure spatiotemporal distributions of gene expression in live tissue explants with a dense extracellular matrix (e.g., tendon). This technology will be ground- breaking for musculoskeletal research since explant models are commonly used to study musculoskeletal tissues and existing techniques requiring cell transfection are not effective due to the dense extracellular matrix.