Assay for detection of early stage apoptosis

摘要早期细胞凋亡的检测在生物医学研究事业的许多领域，特别是在新药和治疗干预措施的开发中具有至关重要的意义。本提案的长期目标是引入一种新的测定方法，用于选择性检测固定组织切片中细胞凋亡的初始阶段。本项目的具体目的是：1)建立一种新的固定组织切片早期细胞凋亡特异性检测方法。2)利用多种细胞凋亡实验模型对新建立的方法进行综合检验。该试验将利用牛痘拓扑异构酶I的独特酶性质来标记II型细胞拓扑异构酶在细胞凋亡初始阶段产生的特征DNA切割。这些双链DNA切割含有末端5'OH基团的4个碱基长的悬垂，目前的技术无法选择性地可视化。新的检测方法将填补在组织切片格式中标记早期细胞凋亡技术的空白。它将具有单细胞敏感性限制，并将使用多种不同的细胞凋亡诱导模型进行测试，包括皮质类固醇，温度，癌症和缺血诱导的细胞凋亡，以及非凋亡性DNA切割。该方法将成为细胞凋亡检测中一个方便实用的工具。它将在早期细胞凋亡检测非常重要的生物医学和新药开发中具有广泛的适用性。拟议的项目将导致开发一种新的化验方法，以满足医疗诊断和病理学的需要。这项技术将简化并降低对细胞死亡和DNA损伤具有预后价值的疾病（如阿尔茨海默病和各种癌症）治疗效果的评估成本。

DESCRIPTION (provided by applicant): Assay for detection of early stage apoptosis Abstract Detection of the early stages of apoptosis is of critical importance in many fields of biomedical research enterprise and especially in the development of the new drugs and therapeutic interventions. The long-term objective of this proposal is to introduce a new assay for the selective detection of the initial stages of apoptosis in fixed tissue sections. Specific Aims of this project are: 1) To develop a new assay for specific detection of early stage apoptosis in fixed tissue sections. 2) To comprehensively test the newly developed assay using multiple experimental models of apoptotic death. The assay will use the unique enzymatic properties of vaccinia topoisomerase I to label characteristic DNA cuts produced by type II cellular topoisomerases in the initial stages of apoptosis. These double-strand DNA cuts contain 4 base-long overhangs with terminal 5'OH groups and cannot be selectively visualized by present techniques. The new assay will fill the gap in techniques for labeling early apoptosis in the tissue section format. It will have a single cell sensitivity limit and will be tested using multiple different models of apoptosis induction including corticosteroid-, temperature-, cancer-, and ischemia- induced apoptosis, as well as non-apoptotic DNA cleavage. The assay will be a convenient and practical tool in the apoptosis detection arsenal. It will have broad applicability in biomedicine and in the development of new drugs where detection of early apoptosis is important. The proposed project will result in the development of a new assay for the needs of medical diagnostics and pathology. The technology will simplify and make it cheaper to evaluate the effects of therapy in diseases where cell death and DNA damage have prognostic value, such as Alzheimer's disease, and various cancers.