Ligatable fluorescent probes using energy transfer for apoptosis detection

项目描述（申请人提供）：本项目将介绍一种新的凋亡细胞原位检测方法。该方法将适用于固定组织样本和活细胞培养。它将检测caspase依赖和caspase非依赖机制驱动的细胞凋亡。该方法将使用荧光能量转移（FET）寡聚探针检测由caspase依赖性和非依赖性凋亡核酸酶产生的末端为3'OH/5'PO4的钝端DNA切割。这些切口是坏死中不存在的细胞凋亡的选择性和普遍性标志。FET探针将通过改变颜色来指示成功检测到它们的目标断裂。这种新的细胞凋亡标记技术将使更灵敏的细胞凋亡检测和区分程序性和非程序性细胞死亡成为可能。本课题的具体目标是：1)利用荧光能量转移探针和T4 DNA连接酶开发一种新的通用的细胞凋亡检测技术，该技术将选择性地标记各种凋亡刽子手核酸酶常见的特定类型的钝端DNA断裂。用几种细胞凋亡和坏死模型在固定组织切片上检测探针。2)将Aim 1中开发的方法扩展到活细胞培养。利用细胞凋亡和坏死培养模型验证不同类型凋亡细胞死亡的选择性标记及其与坏死的区分。3)通过将其应用于局灶性脑缺血作为坏死和多种细胞凋亡同时存在的情况来验证新方法。目的探讨实验性脑卒中后大鼠脑内凋亡和坏死细胞死亡的发生及动态。拟议的研究将引入新的使能技术，对广泛的临床和研究研究很重要。它在脑缺血中的应用将为开发精确有效的治疗干预提供必要的信息。

DESCRIPTION (provided by applicant): This project will introduce a new approach for in situ detection of apoptotic cells. The approach will be applicable to fixed tissue samples and to live cell cultures. It will detect apoptosis driven by caspase- dependent and caspase-independent mechanisms. The approach will use fluorescence energy transfer (FET) oligoprobes to detect blunt ended DNA cuts with terminal 3'OH/5'PO4 produced by caspase-dependent and -independent apoptotic nucleases. These cuts represent a selective and general marker of apoptosis absent in necrosis. The FET probes will indicate successful detection of their target breaks by changing color. This new apoptosis labeling technology will make possible more sensitive apoptosis detection and discrimination between programmed and non-programmed cell death. The Specific Aims of the proposal are: 1) To develop a new and general apoptosis detection technology using fluorescence energy transfer probes and T4 DNA ligase, which will selectively label a specific type of blunt-ended DNA breaks common for various apoptotic executioner nucleases. To test the probes in fixed tissue sections using several models of apoptosis and necrosis. 2) To expand the methodology developed in the Aim 1 to live cell cultures. To verify selective labeling of different types of apoptotic cell death and its discrimination from necrosis using cell culture models of apoptosis and necrosis. 3) To validate the new approach by applying it to study focal cerebral ischemia as a condition where necrosis and several types of apoptosis are simultaneously present. To investigate the initiation and dynamics of apoptotic and necrotic cell death in brain after experimental stroke in rats. The proposed research will introduce the new enabling technology important for a wide range of clinical and research studies. Its application to brain ischemia will provide information essential for the development of precise and 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.