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 胞吞作用——吞噬垂死细胞是一个重要的调节过程。它可以抑制或促进 自我免疫、炎症以及病毒或肿瘤 DNA 的释放。胞吞作用是一种集体细胞 由专业吞噬细胞或周围组织细胞完成的事件。细胞凋亡的早期观点 因为普遍的抗炎过程已经改变。现在分为免疫原性和免疫原性 基于吞噬过程的耐受性（非炎症）形式。因此，有必要拥有一个 广谱技术，可以检测和量化所有参与细胞类型的细胞处理， 然而，目前还没有这样的测定。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新技术，用于检测组织切片和活体中的胞吞作用 细胞。我们的方法分离出独特的酶活性，该活性仅短暂存在于催化循环中 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 拓扑异构化反应的中间体作为新型荧光探针。这些 FRET 探针能够 检测和定量胞吞作用的基本标志物、DNase II 消化 DNA 的溶核活性 凋亡细胞。该技术将选择性地标记和量化细胞凋亡 DNA 分解 胞质体，即吞噬凋亡细胞核的细胞的吞噬溶酶体。新方法将允许 高度特异性的检测以及吞噬细胞和吞噬细胞的近乎瞬时 FRET 定量 活细胞和固定细胞和组织中的反应。 该提案将实现以下具体目标： 1. 开发第一个利用拓扑异构反应催化中间体作为新型原位检测方法 荧光探针检测和量化组织切片格式中的胞吞作用。为了优化特异性， 使用体外和原位模型进行测定的灵敏度和再现性。为了验证新方法 使用组织切片进行细胞清除的几种模型。 2. 开发首个以拓扑异构反应催化中间体为基础的FRET检测技术 新型 FRET 探针，用于在单个细胞水平上标记和定量活细胞中的胞吞作用。到 使用体外、原位模型和活细胞优化测定的特异性、灵敏度和定量能力。 使用培养细胞在几种细胞清除模型中验证新的 FRET 方法。 3. 通过从根本上提高细胞的敏感性来实现胞吞作用效率的亚细胞评估 技术借助新颖的先进荧光标签 - Ndots。优化测定以实现单 分子分辨率评估其分子靶点和广泛的信号量化。最终确定 通过在多种结合细胞凋亡的模型中验证其定量能力来开发该技术 及其细胞清除率。