FRET detection and in situ quantification of efferocytosis using designed enzymatic activity

使用设计的酶活性对胞吞作用进行 FRET 检测和原位定量

• 批准号: 10564789
• 负责人: VLADIMIR V DIDENKO
• 金额: $ 35.42万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-21 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10564789
• 关键词: Anti-Inflammatory Agents; Apoptosis; Apoptotic; Autoimmune Diseases; Biological Assay; Biomedical Research; Biomedical Technology; Cell Nucleus; Cells; Cultured Cells; DNA; Deoxyribonucleases; Detection; Development; Ensure; Event; Excision; Fluorescence Resonance Energy Transfer; Fluorescent Probes; Genetic; Goals; Heart Diseases; Immunization; Immunohistochemistry; In Situ; In Vitro; Individual; Inflammation; Invaded; Label; Malignant Neoplasms; Methods; Modeling; Molecular; Molecular Target; Necrosis; Normal tissue morphology; Nuclear; Organism; Pathology; Pathway interactions; Phagocytes; Phagocytosis; Phagolysosome; Process; Proteins; Reaction; Reproducibility; Research; Resolution; Signal Transduction; Specificity; Stroke; Systemic Lupus Erythematosus; Technology; Tissues; Topoisomerase; Virus Diseases; base; biomedical imaging; cell type; design; immunogenic; macrophage; new technology; novel; single molecule; technology development; tumor DNA; viral DNA

Abstract Efferocytosis - the engulfment of dying cells is an essential regulatory process. It can either inhibit or promote self-immunization, inflammation and the release of viral or tumor DNA. Efferocytosis is a collective cellular event done by either professional phagocytes or by the surrounding tissue cells. The earlier view of apoptosis as a universally anti-inflammatory process has changed. It is now divided into the immunogenic and the tolerogenic (non-inflammatory) forms based the engulfment process. Therefore it became essential to have a broad-spectrum technology which could detect and quantify efferocytic processing in all participating cell types, However, currently there is no such assay. In this project we set out to overcome this limitation. This project will introduce a principally new technology detecting efferocytosis in tissue sections and in live cells. Our method isolates the unique enzymatic activity, present only transiently in the catalytic cycle of topoisomerases, and uses it as a basis for the new type of molecular labeling. The assay will use catalytic intermediates of the topoisomerization reaction as novel fluorescent probes. These FRET probes are capable of detecting and quantifying the essential marker of efferocytosis, nucleolytic activity of DNase II digesting DNA of apoptotic cells. The technology will selectively label and quantify apoptotic DNA disassembly in efferosomes, i.e. phagolysosomes of the cells that engulfed apoptotic nuclei. The new method will permit highly specific detection and the near instantaneous FRET quantification of efferocytic and phagocytic reactions in live and fixed cells and tissues. The proposal will reach these Specific Aims: 1. To develop the first in situ assay using catalytic intermediates of topoisomerization reaction as novel fluorescent probes detecting and quantifying efferocytosis in the tissue section format. To optimize specificity, sensitivity and reproducibility of the assay using in vitro and in situ models. To validate the new method in several models of efferocytic clearance using tissue sections. 2. To develop the first FRET assay technology using catalytic intermediates of topoisomerization reaction as novel FRET probes for labeling and quantification of efferocytosis in live cells at the level of individual cells. To optimize specificity, sensitivity and quantitative ability of the assay using in vitro, in situ models, and live cells. To validate the new FRET method in several models of efferocytic clearance using cultured cells. 3. To enable the subcellular assessment of efferocytosis efficiency by radically increasing sensitivity of the technology with help of novel advanced fluorescent tags - Ndots. To optimize the assay to achieve single molecule resolution in assessing its molecular targets and in broad range signal quantification. To finalize development of the technology by validating its quantification capacity in several models combining apoptosis and its efferocytic clearance.

抽象的 吞噬作用 - 死亡细胞的吞噬是必不可少的调节过程。它可以抑制或促进 自免疫，炎症和病毒或肿瘤DNA的释放。胚细胞增多症是一种集体细胞 由专业吞噬细胞或周围组织细胞完成的事件。凋亡的早期观点 由于普遍的抗炎过程已经改变。现在它分为免疫原性和 基于吞噬过程的耐受性（非炎症）形式。因此，拥有一个 广谱技术可以检测和量化所有参与细胞类型中的胚细胞处理， 但是，目前没有这样的测定法。在这个项目中，我们着手克服这一限制。 该项目将引入一项主要的新技术，以检测组织段和现场的肿瘤病 细胞。我们的方法分离了独特的酶活性，仅在 拓扑异构酶，并将其用作新型分子标记的基础。该测定将使用催化 拓扑异构反应的中间体作为新的荧光探针。这些FRET探针能够 检测和量化常见性吞噬作用的基本标志，DNase II消化DNA的核酸化活性 凋亡细胞。该技术将选择性地标记并量化凋亡DNA拆卸 福音小体，即吞噬凋亡核的细胞的吞噬体。新方法将允许 高度特异性的检测以及近瞬时的FRET定量递增细胞和吞噬细胞 活细胞和组织中的反应。 该提案将达到这些具体目标： 1。使用拓扑异构反应的催化中间体开发第一个原位测定 荧光探针检测和量化组织截面格式中的传染性细胞增多症。为了优化特异性， 使用体外和原位模型的测定的灵敏度和可重复性。验证新方法 使用组织切片的几种胚细胞清除模型。 2。使用拓扑异构化反应的催化中间体开发第一个FRET分析技术 新型的FRET探针，用于在单个细胞水平上在活细胞中标记和定量吞噬作用。到 优化使用体外，原位模型和活细胞的测定的特异性，灵敏度和定量能力。 在使用培养细胞的几种模型中验证新的FRET方法。 3。通过从根本上提高敏感性来实现肿瘤效率的亚细胞评估 在新型的高级荧光标签-NDOTS的帮助下进行技术。优化实现单一的测定法 分子在评估其分子靶标和广泛信号定量时的分辨率。最终确定 通过验证凋亡结合的多种模型中的量化能力来开发该技术 及其胚细胞清除率。