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

使用条形码生物传感器对活体肌腱外植体中的基因表达和微型组织菌株进行共定位

• 批准号: 10373315
• 负责人: Spencer Szczesny
• 金额: $ 19.91万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10373315
• 关键词: Address; Algorithms; Bar Codes; Biophysics; Bioreactors; Biosensor; Biotechnology; Calcium; Catabolism; Cell Communication; Cell Nucleus; Cells; Chronic; Collagen Fibril; Degenerative Disorder; Deposition; Development; Devices; Diffuse; Disease; Endocytosis; Environment; Extracellular Matrix; Fatigue; Fatty acid glycerol esters; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescent in Situ Hybridization; Gene Expression; Genes; Goals; Gold; Image; In Vitro; Inflammatory; Interleukin-1 beta; Knowledge; Label; Maps; Matrix Metalloproteinases; Measurement; Measures; Mechanics; Mediating; Messenger RNA; Microscope; Modeling; Mus; Musculoskeletal; Oligonucleotide Probes; PPAR gamma; PTGS2 gene; Peptide Hydrolases; Persistent pain; Proteins; Protocols documentation; Reporter; Research; Risk Factors; Scheme; Signal Transduction; Spatial Distribution; Stimulus; Structure; Techniques; Technology; Tendinopathy; Tendon structure; Testing; Time; Tissues; Transfection; Transgenic Mice; Weight-Bearing state; achilles tendon; cartilaginous; collagenase 3; cost; cytokine; density; design; experimental study; fluorophore; in vivo; inflammatory marker; innovation; interest; locked nucleic acid; mechanical load; mechanical stimulus; mechanotransduction; nanorod; novel therapeutics; optical spectra; prevent; repaired; response; spatiotemporal; tendon rupture; two-dimensional

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.

项目概要 肌腱病是一种慢性退行性疾病，是由于肌腱过度使用（即疲劳负荷）而发生的。 除了机械损伤之外，肌腱疲劳负荷还会诱导分解代谢蛋白酶的表达， 炎症细胞因子以及异常基质成分（例如软骨、脂肪和 钙沉积），进一步削弱组织并推动退化的进展。理解 为什么肌腱细胞无法恢复组织结构反而加剧退化对于预防至关重要 肌腱病。普遍的假设是，细胞对肌腱过度使用的反应适得其反 来自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，有 根本缺乏有关天然组织中肌腱细胞机械转导的知识 环境以及生物物理刺激的改变是否会导致肌腱退化。到 为了解决这一知识差距，该提案的目标是开发一种活组织外植体模型，使 局部机械刺激和细胞基因表达的同步时空测量 肌腱疲劳载荷。通过将肌腱细胞反应与天然存在的机械刺激共定位 细胞微环境，我们可以直接测试机械转导是否是造成负面影响的原因 观察到肌腱退化的细胞反应。实现这一目标的关键技术障碍 目标是开发一种可以动态检测细胞基因空间分布的生物传感器 肌腱外植体中的表达。内源荧光报告基因受到生成时间和成本的限制 具有特定感兴趣基因的转基因小鼠。替代生物技术需要细胞转染，即 在细胞外基质致密的组织中效率低下。因此，我们将研究金纳米棒是否 通过内吞作用内化，可以传递荧光标记的寡核苷酸探针来检测细胞 肌腱外植体中的基因表达。在目标1中，我们将优化金纳米棒锁定核酸的设计 生物传感器并验证其在活体中测量机械敏感基因的灵敏度和空间精度 肌腱外植体。此外，我们将建立一个带有 FRET 探针和光谱的条形码策略 分解算法可同时测量多达十个目标基因。在目标 2 中，我们将使用这项技术 确定对肌腱疲劳的退行性细胞反应是否与肌腱的变化在空间上相关 局部机械刺激（即应变）。这个项目是创新的，因为我们的结果将识别刺激 介导对肌腱过度使用的负面细胞反应，并帮助开发预防肌腱过度使用的新疗法 退化。此外，我们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）的活组织外植体中表达。这项技术将落地- 由于外植体模型通常用于研究肌肉骨骼，因此突破了肌肉骨骼研究 由于细胞外基质致密，需要细胞转染的组织和现有技术并不有效。