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

摘要 效应细胞吞噬死亡的细胞是一个重要的调节过程。它既可以抑制，也可以促进 自身免疫、炎症和病毒或肿瘤DNA的释放。效应细胞增多症是一种集体细胞 由专业吞噬细胞或由周围组织细胞完成的事件。关于细胞凋亡的早期观点 作为一个普遍的消炎过程已经发生了变化。它现在分为免疫原性和 基于吞噬过程的耐受性(非炎性)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的泡沫化过程， 然而，目前还没有这样的检测方法。在这个项目中，我们着手克服这一限制。 该项目将引入一种主要的新技术，检测组织切片和活体中的泡沫化。 细胞。我们的方法分离出独特的酶活性，只在催化循环中短暂存在 拓扑异构酶，并将其作为新型分子标记的基础。化验将使用催化 拓扑异构化反应中间体作为新型荧光探针。这些FRET探头有能力 检测和定量泡沫化的基本标志--DNase II消化DNA的溶核活性 凋亡细胞的数量。这项技术将选择性地标记和量化细胞中的凋亡DNA分解 泡沫体，即吞噬凋亡细胞核的细胞的吞噬溶酶体。新方法将允许 泡泡细胞和吞噬细胞的高特异性检测和近瞬时FRET定量 在活的和固定的细胞和组织中的反应。 这项建议将达到以下具体目标： 1.首次将拓扑异构化反应的催化中间体用于原位检测 荧光探针检测和量化组织切片格式的胞吐作用。为了优化专一性， 体外模型和原位模型检测的灵敏度和重复性。要在中验证新方法 使用组织切片的几种模型的泡出细胞清除。 2.首次建立了以拓扑异构化反应催化中间体为基础的FRET检测技术 新的FRET探针用于在单个细胞水平上标记和定量活细胞中的胞吐作用。至 使用体外、原位模型和活细胞优化检测的特异性、敏感性和定量能力。 用培养的细胞验证新的FRET方法在几种胞外清除模型中的有效性。 3.通过从根本上增加泡沫吞噬效率的敏感性来实现亚细胞评估 在新型先进荧光标签--NDOTS的帮助下，我们采用了先进的技术。优化检测方法，实现单项检测 在评估其分子靶标时的分子分辨率和在宽范围内的信号量化。最后敲定 通过在几种结合细胞凋亡的模型中验证其量化能力来开发该技术 以及它的出泡细胞清除量。

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.