Ligatable fluorescent probes using energy transfer for apoptosis detection

描述（由申请人提供）：本项目将介绍一种原位检测凋亡细胞的新方法。该方法将适用于固定的组织样品和活细胞培养物。它将检测由半胱天冬酶依赖性和半胱天冬酶非依赖性机制驱动的细胞凋亡。该方法将使用荧光能量转移（FET）寡聚探针来检测由半胱天冬酶依赖性和非依赖性凋亡核酸酶产生的末端3 'OH/5' PO 4的平端DNA切割。这些切口代表了坏死中不存在的细胞凋亡的选择性和一般性标记。FET探头将通过改变颜色指示成功检测到其目标断裂。这种新的凋亡标记技术将使更灵敏的凋亡检测和程序性和非程序性细胞死亡之间的区别成为可能。1）利用荧光能量转移探针和T4 DNA连接酶，建立一种新的、通用的凋亡检测技术，该技术将选择性地标记一种特定类型的平末端DNA断裂，该断裂是各种凋亡执行核酸酶所共有的。使用几种细胞凋亡和坏死模型在固定的组织切片中测试探针。2)将目标1中开发的方法扩展到活细胞培养。使用细胞凋亡和坏死的细胞培养模型验证不同类型的凋亡细胞死亡的选择性标记及其与坏死的区分。3)通过将其应用于研究局灶性脑缺血作为同时存在坏死和几种类型的凋亡的条件来验证新方法。探讨实验性脑卒中后脑组织细胞凋亡和坏死的发生及动态变化。拟议的研究将引入新的使能技术，这对广泛的临床和研究很重要。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。 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): 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.