一种研究和鉴别悬浮培养红豆杉细胞凋亡与坏死的新方法

A Novel Method for Investigation and Differentiation Between Apoptosis and Necrosis of Plant Cells in Suspension Culture

将在动物细胞凋亡研究中应用的Hoechst PI双重荧光染色法与琥珀酸脱氢酶 (SDH)染色法相结合 ,建立了一种更加完善的、能同时鉴别和研究悬浮培养的植物细胞凋亡及坏死的新方法———Hoechst PI SDH三重染色法 (H P S法 )。该方法可直接用于红豆杉悬浮培养细胞 ,无需对细胞进行去壁、固定及切片等其它方法所必需的步骤 ,在荧光显微镜下可鉴别活细胞、死细胞及凋亡细胞 ,并可同时观测细胞凋亡的全部过程。该方法简单、快速、准确 ,而且克服了因细胞膜通透性差异引起的对死、活细胞判断的困难 ,可在植物细胞凋亡的研究中广泛应用。

A novel method, which is more reliable and convenient in detecting cells undergoing apoptosis, and can distinguish them from those cells undergoing necrosis, is developed. The method, termed H-P-S triple-staining, is based on the combination of Hoechst33342-propidium iodide(PI) double-fluoresence stain method, which is usually applied to studies of apoptosis of animal cells, with histochemical staining of succinate dehydrogenase(SDH). The plant cells under study can be stained and examined without being propared to protoplasts and those undergoing apoptosis can be detected. This method is highly discriminating between cells undergoing apoptosis and those undergoing necrosis. In addition, the live cells, cells undergoing apoptosis and those undergoing necrosis are differentiated simultaneously under a fluorescence microscopy and the whole process and the typical morphological features of apoptosis are also observed. The results demonstrate that this method is simpler, quicker and more accurate; moreover, the method overcomes the difficulty of discriminating the cells undergoing apoptosis from the cells undergoing necrosis by the alteration of the cell membrane permeability. Thus, this method is useful in studying on apoptosis of the plant cells.