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How do the primary cultures of human umblical endothelium become resistant to vero toxins ?

人类脐带内皮细胞的原代培养物如何对维罗毒素产生抵抗力？

基本信息

批准号：
10670274
负责人：
YOSHIDA Tomoaki
金额：
$ 1.66万
依托单位：
Aichi Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

The endothelial cell damage by Shiga toxin (Stx) -1 / -2, both of which are produced by enterohemorrhagic E. coli, has been believed to the crucial for the pathogenesis of hemolytic uremic syndrome. In in vitro systems, however, Stxs were hardly toxic against various human endothelial cells such as umbilical vein endothelial cells (HUVEC). To our surprise, the fresh primary cultures of HUVEC, appeared to be sensitive to merely pM order of Stxs, whereas they rendered resistant during passage of only 1-2 weeks. Moreover, apoptosis is apparently involved in the damage of the primary culture cells, but not of passaged ones, as shown by the annexin V stain, TUNEL or DNA fragmentation, despite that the well-established function of Stxs is the inhibition of protein synthesis. Concerning the intracellular event in apoptosis, the activation of caspase -3 was observed by immunoblotting, and correspondingly, the inhibitor of caspase-3 raised the viability of endothelial cells after Stx treatment. Common upstream cascades for apoptosis such as fas stimulation, mitochondria discharge and nitric oxide synthesis were examined and found not be engaged, indicating a novel unique path may be at work here. Although the mechanism underling the resistance of the passaged HUVEC is still unclear, the possible candidates of extracellular and intracellular factors may be IFN-αand bcl-xL, respectively, according to our data

肠出血性大肠杆菌产生的志贺毒素(STX)-1/-2对血管内皮细胞的损伤被认为是溶血性尿毒症综合征发病的关键。然而，在体外系统中，STX对各种人内皮细胞，如脐静脉内皮细胞(HUVEC)几乎没有毒性。令我们惊讶的是，HUVEC的新鲜原代培养物似乎仅对Pm顺序的STX敏感，而它们在传代仅1-2周时就产生了抗性。此外，尽管Stxs的功能是抑制蛋白质合成，但通过Annexin V染色、TUNEL或DNA片段化显示，细胞凋亡明显参与了原代培养细胞的损伤，而不是传代细胞的损伤。对于细胞内的凋亡事件，免疫印迹法观察到caspase-3的激活，相应地，caspase-3的抑制剂提高了STX处理后内皮细胞的活力。对Fas刺激、线粒体放电和一氧化氮合成等常见的上游凋亡级联反应进行了研究，发现它们不参与其中，表明一条新的独特途径可能在这里发挥作用。尽管传代的人脐静脉内皮细胞耐药的机制尚不清楚，但根据我们的数据，细胞外和细胞内因子可能分别是干扰素-α和bcl-xl。