Li Can, Ran Xueqin, Wang Jiafu
School of Animal Science, Guizhou University, Guiyang 550025, China.
Wei Sheng Wu Xue Bao. 2009 May;49(5):658-63.
Shiga-like toxin-producing Escherichia coli (STEC) causes edema disease in piglets and hemolytic uremic syndrome in human. Shiga-like toxins (Stxs) produced by STEC induce mammalian cells death via either necrosis or apoptosis. However, the ability of stx2e, separated from edema disease (Stx2e), to trigger apoptosis and the sequence of intracellular signaling events have not yet been completely defined. In this study we investigated the apoptotic effects of Stx2e on Vero cells.
Vero cells were treated with different concentrations of Stx2e for different time and the apoptotic cells were characterized by acridine orange and ethidium bromide fluorescent dye staining. The fragmentation of chromatin from Vero cells treated with Stx2e were detected by agarose gel electrophoresis. The expression patterns of apoptosis-associated factors were assayed by Western blotting.
Stx2e-treated cells showed characteristic features of apoptosis, including membrane blebbing, DNA fragmentation, chromatin condensation, and the formation of apoptotic bodies, whereas ricin did not induce apoptosis of Vero cells even at a high dose. Fluorescent dye staining showed that Stx2e induced apoptosis of Vero cells in dose- and time-dependent manners. Caspase-3 was activated whereas expression levels of bcl2 associated X protein (Bax) and caspase-9 had no change compared with the negative control.
Stx2e induced intensively apoptosis of Vero cells, which was mediated through the mitochondrion-independent pathway and might be throught a receptor-dependent pathway.
产志贺样毒素大肠杆菌（STEC）可导致仔猪水肿病和人类溶血尿毒综合征。STEC产生的志贺样毒素（Stxs）可通过坏死或凋亡诱导哺乳动物细胞死亡。然而，从水肿病中分离出的stx2e触发细胞凋亡的能力以及细胞内信号事件的顺序尚未完全明确。在本研究中，我们调查了Stx2e对Vero细胞的凋亡作用。
用不同浓度的Stx2e处理Vero细胞不同时间，并用吖啶橙和溴化乙锭荧光染料染色来鉴定凋亡细胞。通过琼脂糖凝胶电泳检测用Stx2e处理的Vero细胞的染色质片段化。通过蛋白质免疫印迹法检测凋亡相关因子的表达模式。
经Stx2e处理的细胞呈现出凋亡的特征，包括细胞膜起泡、DNA片段化、染色质浓缩以及凋亡小体的形成，而蓖麻毒素即使在高剂量下也不诱导Vero细胞凋亡。荧光染料染色表明，Stx2e以剂量和时间依赖性方式诱导Vero细胞凋亡。与阴性对照相比，半胱天冬酶-3被激活，而bcl2相关X蛋白（Bax）和半胱天冬酶-9的表达水平没有变化。
Stx2e强烈诱导Vero细胞凋亡，这是通过不依赖线粒体的途径介导的，可能是通过受体依赖性途径。
