Ligatable fluorescent probes using energy transfer for apoptosis detection

用于细胞凋亡检测的使用能量转移的可连接荧光探针 摘要 本项目将介绍一种原位检测凋亡细胞的新方法。该办法 适用于固定的组织样品和活细胞培养物。它会检测到由半胱天冬酶驱动的细胞凋亡- 依赖性和半胱天冬酶非依赖性机制。该方法将使用荧光能量 转移（FET）寡探针，以检测具有末端3 'OH/5' PO 4的平端DNA切割，所述末端3 'OH/5' PO 4由 半胱天冬酶依赖性和非依赖性凋亡核酸酶。这些削减代表了一种选择性的， 凋亡的一般标志物在坏死中缺失。FET探针将指示成功检测到 他们的目标会通过改变颜色来破坏这种新的凋亡标记技术将使更多的 灵敏的凋亡检测和程序化与非程序化细胞的区分 死亡该提案的具体目标是： 1)建立一种新的、通用的细胞凋亡荧光能量转移检测技术 探针和T4 DNA连接酶，将选择性标记特定类型的平端DNA断裂 常见于各种凋亡执行者核酸酶。使用以下工具在固定组织切片中测试探头 细胞凋亡和坏死的几种模型。 2)将目标1中开发的方法扩展到活细胞培养。验证选择性标记 不同类型的凋亡细胞死亡和它的区别坏死使用细胞培养模型， 凋亡和坏死。 3)通过将其应用于研究局灶性脑缺血来验证新方法， 同时存在坏死和几种类型的凋亡。调查入会仪式， 大鼠实验性脑卒中后脑细胞凋亡和坏死的动态变化 拟议的研究将引入新的使能技术，这对广泛的临床应用非常重要。 和研究。它在脑缺血中的应用将为脑缺血的研究提供必要的信息。 制定精确有效的治疗干预措施。

Ligatable fluorescent probes using energy transfer for apoptosis detection Abstract 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.