利用核磁共振技术检测植物细胞凋亡

在细胞凋亡的研究中,通常以检测细胞核和细胞器的形态改变或生物化学特性变化为指标进行分析。已有的实验表明: 动物细胞在凋亡过程中, 细胞膜的脂质双分子层发生了一系列生物物理和生物化学改变,如膜电位的改变、磷脂酰丝氨酸由细胞膜内侧向外翻转、细胞膜微粘度的改变等,这些变化会导致细胞中亚甲基信号强度的增加。我们利用质子核磁共振光谱分析(1H-NMR)方法, 首次发现用物理和化学方法诱导的烟草(Nicotiana tabacumL. cv. BY-2)和胡萝卜(Daucus carota L.)细胞在凋亡过程中伴随有亚甲基信号强度的明显增加。在用烟酰胺处理的烟草细胞中, 亚甲基信号强度的增加与DNA Ladder 几乎同时出现,随诱导时间的延长, 亚甲基信号强度也逐渐增大,在24 h亚甲基信号强度增加约2倍。而这种特征在坏死的细胞中并不存在。说明亚甲基信号强度的增加是动、植物细胞凋亡过程中所具有的共同特征,1H-NMR技术提供了一种精确可靠的分析植物细胞凋亡的手段,同时由于它所具有的非侵害性的特点,可能在揭示细胞凋亡机制的研究中具有一定的意义。

Increase in Methylene Resonance Signal Intensity Is Associated with Apoptosis in Plant Cells as Detected by ~1H-NMR

Apoptosis, both in animal and plant cells, is usually analyzed by observing and detecting thecharacteristic morphological and biochemical changes in nuclei, organelles such as mitochondria and cellmembranes. Using 1H-NMR we have found a correlation between the progression of apoptosis and 1H-NMRmethylene signal intensity in cultured tobacco (Nicotiana tabacum L. cv. BY-2) and carrot (Daucus carotaL.) cells. The increase in the methylene resonance signal intensity which reflects the alterations inmembrane lipids and accordingly in the biophysical and biochemical characteristics of cell membraneduring apoptosis was found to be closely associated with nuclear changes as detected by DNA ladderingassay and the TUNEL procedure. The rise of methylene resonance signal intensity was evident in apoptoticplant cells induced by various factors including nicotinamide, menadione, heat shock and hydroxyl radicals,but it was not found in necrotic cells. To our knowledge, this is the first report of the 1H-NMR detection ofapoptosis which is closely associated with the increase in methylene resonance signal intensity in plants.