Wu Yi-Chun, Wang Xiaochen, Xue Ding
Institutes of Molecular and Cellular Biology, National Taiwan University, Taipei 10617, Taiwan.
Methods Cell Biol. 2012;107:295-320. doi: 10.1016/B978-0-12-394620-1.00010-2.
Programmed cell death or apoptosis is a common cell fate during the development of multicellular organisms, including C. elegans. C. elegans has been proven to be an excellent model organism for studies of programmed cell death. Its transparency and the knowledge of its cell lineage, including its invariant life vs. death fate of all cells, allow programmed cell death to be studied in vivo at single cell resolution in a way that no other systems can currently match. This advantage, in combination with sophisticated genetic manipulations, has facilitated the identification and characterization of many genes important for different aspects of programmed cell death. In addition to the ability to observe life and death of cells directly under Nomarski optics or by using fluorescent transgene products in living animals, several methods have been developed to study other aspects of apoptosis. For example, fragmentation of chromosomal DNA and the identity of phagocytic cells that engulf cell corpses can be analyzed using specific staining methods. Proteases and nucleases involved in cell killing and DNA degradation, respectively, can be studied using specific biochemical assays. These cell death assays have been instrumental in elucidating the functions of cell death-related genes and the mechanisms by which they affect cell death. In this chapter, we will review and describe these methods in detail.
程序性细胞死亡或凋亡是多细胞生物（包括秀丽隐杆线虫）发育过程中常见的细胞命运。秀丽隐杆线虫已被证明是研究程序性细胞死亡的优秀模式生物。其透明度以及细胞谱系的相关知识，包括所有细胞固定的生死命运，使得程序性细胞死亡能够在体内以单细胞分辨率进行研究，这是目前其他系统无法比拟的。这种优势与复杂的基因操作相结合，有助于鉴定和表征许多对程序性细胞死亡不同方面重要的基因。除了能够在Nomarski光学显微镜下直接观察活细胞中的生死情况或通过使用荧光转基因产物进行观察外，还开发了几种方法来研究凋亡的其他方面。例如，可使用特定染色方法分析染色体DNA的片段化以及吞噬细胞尸体的吞噬细胞的身份。可分别使用特定的生化测定法研究参与细胞杀伤和DNA降解的蛋白酶和核酸酶。这些细胞死亡检测方法有助于阐明细胞死亡相关基因的功能及其影响细胞死亡的机制。在本章中，我们将详细回顾和描述这些方法。
