Assay for dual detection of apoptosis

描述（由申请人提供）：细胞凋亡是一个有序的细胞消除过程，它确保受损细胞的完全解体和消失。最近，凋亡细胞消除的概念被扩展，程序性细胞死亡不再被视为单个细胞事件。完整的凋亡过程现在包括两个阶段：自我驱动的细胞解体和吞噬细胞对凋亡细胞尸体的外部控制消除。第二吞噬阶段是必不可少的，高度保守的，被认为比细胞解体的内部阶段更重要。这是因为它确保了垂死细胞DNA的完全降解，防止了病理、病毒和肿瘤DNA的释放和自我免疫。在从哺乳动物到苍蝇的不同细胞和物种中，只有两组独立且保守的酶负责在细胞凋亡的第一和第二阶段消除细胞DNA。产生DNase I型DNA切割的核酸酶对于细胞凋亡执行的第一阶段是必不可少的，而单一的酶DNase II在细胞凋亡的“扫荡”阶段中起着重要作用。可以检测两个酶组的活性的探针将允许全面标记凋亡执行。在这个项目中，我们将介绍这样一种方法。我们的技术利用了这两种类型的核酸酶产生可识别的签名DNA断裂的事实。新的检测方法将同时检测组织切片中凋亡DNA降解的细胞内和吞噬阶段。它是基于多重标记的签名DNA切割产生的刽子手和吞噬核酸酶。本研究的具体目的是：1）建立一种双重检测的凋亡检测方法，用于标记凋亡细胞解体的内部和吞噬阶段; 2）在永久性局灶性脑缺血模型中测试新技术。新的检测方法将是一个独特的和实用的工具，在细胞凋亡检测武库。它将在生物医学中具有广泛的应用。其应用于脑缺血将提供有用的信息，为有效的治疗干预措施的发展。公共卫生相关性：拟议项目将导致开发一种新的测定方法，以满足医学诊断和病理学的需求。该技术将允许精确评估细胞死亡和DNA损伤具有预后价值的疾病的治疗效果，如中风，阿尔茨海默病和各种癌症。

DESCRIPTION (provided by applicant): Apoptosis is an orderly process of cellular elimination, which ensures complete disassembly and disappearance of damaged cells. Recently the concept of apoptotic cell elimination was expanded and programmed cell death is no longer viewed as an individual cellular event. The complete apoptotic process now includes two phases: the self-driven cell disassembly and externally-controlled elimination of apoptotic cell corpses by phagocytizing cells. The second phagocytic phase is essential, highly conserved and is considered to be even more important than the internal phase of cell disassembly. This is because it ensures the complete degradation of the dying cell's DNA, preventing release of pathological, viral and tumor DNA and self-immunization. In different cells and species from mammals to flies, only two separate and conserved groups of enzymes are responsible for elimination of cellular DNA in the first and second phases of apoptosis. The nucleases producing DNase I-type DNA cleavage are essential for the first phase of apoptotic execution, whereas a single enzyme DNase II plays a fundamental role in the 'mopping up' phase of apoptosis. The probes which could detect activity of both enzymatic groups would permit comprehensive labeling of apoptotic execution. In this project we will introduce such an approach. Our technology capitalizes on the fact that both types of nucleases produce identifiable signature DNA breaks. The new assay will simultaneously detect both the intracellular and phagocytic phases of apoptotic DNA degradation in tissue sections. It is based on multi-labeling of signature DNA cuts produced by the executioner and phagocytic nucleases. The Specific Aims of the proposal are: 1) To develop a dual-detection apoptotic assay for labeling of internal and phagocytic stages of apoptotic cell disassembly; 2) To test the new technology in the model of permanent focal brain ischemia. The new assay will be a unique and practical tool in the apoptosis detection arsenal. It will have broad applicability in biomedicine. Its application to brain ischemia will provide information useful for the development of effective therapeutic interventions. PUBLIC HEALTH RELEVANCE: The proposed project will result in the development of a new assay for the needs of medical diagnostics and pathology. The technology will allow precise evaluation of the effects of therapy in diseases where cell death and DNA damage have prognostic value, such as stroke, Alzheimer's disease, and various cancers.