FRET probes for analysis of serpin-mediated apoptosis in focal brain ischemia

描述(申请人提供)：FRET探针用于分析局灶性脑缺血中丝氨酸介导的细胞凋亡。鉴于细胞凋亡研究的深远意义，S有必要有能够区分不同凋亡途径的探针。丝氨酸蛋白酶依赖途径是最近发现的最新的凋亡程序之一，但仍缺乏特定的工具来进行原位可视化。Serpin 1B是这一途径的关键成员，是神经组织中细胞凋亡死亡的重要效应因子。在代谢压力下，它会转化为一种活性的核酸内切酶。这种独特的转变发生在低氧条件下，同时暴露出一个活跃的DNA酶位点和一个核定位信号。新形成的DNA降解酶迅速进入细胞核，导致细胞死亡。尽管这种非caspase的凋亡机制非常重要，但目前还没有方法在组织切片和活细胞培养中选择性地标记它。在这个项目中，我们将开发这样的方法，并将它们应用于研究局灶性脑缺血中蛇毒蛋白介导的细胞凋亡。具体目的：1.建立一种新的基于FRET的选择性标记组织切片中丝氨酸介导的细胞凋亡的方法。该方法将同时共同检测丝氨酸1B衍生的核酸内切酶蛋白及其产生的特有的DNA断裂。2.建立一种基于FRET的新方法，用于选择性标记活细胞培养中蛇毒蛋白介导的细胞凋亡。FRET探针只有在检测到丝氨酸介导的途径的特定标记物后才会变得荧光。3.应用新发展的分子工具对局灶性脑缺血进行研究。探讨丝氨酸介导的细胞凋亡在大鼠局灶性脑缺血模型中的作用，探讨其启动机制、动力学和分布规律。该项目将为一般的细胞凋亡研究，特别是对缺血的研究引入使能技术。它们首次系统地应用于局灶性脑缺血，将为今后卒中的临床和研究提供有用的信息，并为开发有效的治疗干预措施提供信息。

DESCRIPTION (provided by applicant): FRET probes for analysis of serpin-mediated apoptosis in focal brain ischemia. In light of the profound significance of apoptosis research, it s essential to have probes which can discriminate between different apoptotic pathways. The serine protease- dependent pathway is one of the most recently identified apoptotic programs, which still lacks specific tools for its visualization in situ. Serpin 1B is a key member of this pathway and is an important effector of apoptotic death in neural tissue. In metabolic stress it transforms into an active endonuclease. This unique transition occurs in hypoxia and simultaneously exposes an active DNase site and a nuclear localization signal. The newly formed DNA degrading enzyme promptly moves into the nucleus causing cell death. In spite of the high importance of this non-caspase apoptosis mechanism, there are currently no methods to selectively label it in tissue sections and in live cell cultures. In this project we will develp such methods and will apply them to study serpin-mediated apoptosis in focal brain ischemia. Specific aims: 1. To develop the new FRET-based approach for selective labeling of serpin-mediated apoptosis in tissue sections. The approach will simultaneously co-detect serpin 1B-derived endonuclease protein and characteristic DNA breaks which it produces. 2. To develop the new FRET-based approach for selective labeling of serpin-mediated apoptosis in live cell cultures. The FRET probes will become fluorescent only after they detect a specific marker of serpin-mediated pathway. 3. To apply the newly developed molecular tools to study focal cerebral ischemia. To evaluate the role of serpin-mediated apoptosis in focal brain ischemia in the rat model; to investigate its initiation mechanisms, dynamics, and patterns of distribution. The project will introduce enabling technologies for apoptosis research in general, and for studies in ischemia in particular. Their first systematic application to focal brain ischemia will provide information useful for the future clinical and research investigations of stroke, and for the development of effective therapeutic interventions.