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

肠出血性大肠杆菌产生的滋贺毒素（Stx）-1 /-2对内皮细胞的损伤作用。大肠埃希菌是溶血性尿毒综合征的致病菌。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级的Stx敏感，而它们仅在1-2周的传代期间就呈现抗性。此外，细胞凋亡显然涉及原代培养细胞的损伤，但不是传代细胞的损伤，如膜联蛋白V染色、TUNEL或DNA片段化所示，尽管Stxs的公认功能是抑制蛋白质合成。关于细胞凋亡中的细胞内事件，通过免疫印迹观察到caspase-3的活化，并且相应地，caspase-3的抑制剂提高了Stx处理后的内皮细胞的活力。细胞凋亡的常见上游级联反应，如fas刺激，线粒体放电和一氧化氮合成被检查，发现没有参与，表明一个新的独特的途径可能在这里工作。虽然传代后HUVEC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的可能候选者分别是IFN-α和bcl-xL

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