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

摘要 胞饮作用-吞噬垂死细胞是一个重要的调节过程。它可以抑制或促进 自身免疫、炎症和病毒或肿瘤DNA的释放。胞吐作用是一种集体的细胞 由专职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 作为一个普遍的抗炎过程已经改变。它现在被分为免疫原性的和 致耐受性（非炎性）形式基于吞噬过程。因此，有必要有一个 广谱技术可以检测和量化所有参与细胞类型中的单核细胞加工， 然而，目前没有这样的测定。在这个项目中，我们着手克服这一限制。 本项目将介绍一种在组织切片和活体中检测红细胞增多症的新技术。 细胞我们的方法分离了独特的酶活性，仅在催化循环中短暂存在， 拓扑异构酶，并将其用作新型分子标记的基础。该分析将使用催化剂 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 检测和定量红细胞增多症的基本标志物，DNA酶II消化DNA的溶核活性 凋亡的细胞。该技术将选择性地标记和定量细胞凋亡DNA分解， 吞噬体，即吞噬凋亡细胞核的细胞的吞噬溶酶体。新方法将允许 高特异性检测和近瞬时FRET定量的巨噬细胞和吞噬细胞 活的和固定的细胞和组织中的反应。 该提案将达到这些具体目标： 1.开发了第一个使用拓扑异构化反应催化中间体的原位分析方法， 荧光探针检测和定量组织切片形式中的红细胞增多症。为了优化特异性， 使用体外和原位模型测定的灵敏度和再现性。中验证新方法 几种使用组织切片的巨噬细胞清除模型。 2.建立了第一个以拓扑异构化反应的催化中间体为模板的FRET检测技术 用于在单个细胞水平上标记和定量活细胞中胞浆细胞增多的新型FRET探针。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。 在几种使用培养细胞的单核细胞清除模型中验证新的FRET方法。 3.为了能够通过从根本上提高细胞因子的灵敏度来亚细胞评估红细胞生成效率， 技术的帮助下，新的先进的荧光标记- Ndots。优化测定，以实现单一 分子分辨率在评估其分子靶标和宽范围信号定量中的应用。最后确定 通过在几种模型中验证其定量能力来开发该技术， 和其巨噬细胞清除率。

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.