Rickettsia rickettsii infection of the EA.hy 926 endothelial cell line:: morphological response to infection and evidence for oxidative injury
Rickettsia rickettsii infection of the EA.hy 926 endothelial cell line:: morphological response to infection and evidence for oxidative injury
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DOI：
10.1099/00221287-144-8-2037
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发表时间：
1998-08-01
期刊：
影响因子：
2.8
通讯作者：
Silverman, DJ
中科院分区：
文献类型：
--
作者：
Eremeeva, ME;Silverman, DJ
eah926是一种表达人类血管内皮高度分化功能的永久性细胞系。立克次体能有效感染eahi926细胞并引起细胞病变。在第2天应用含有2 μ g艾美汀ml（-1）和40 μ g NaF ml（-1）的二次琼脂糖覆盖层后，立克次体在感染后10天（p.i）在eah .hy 926细胞中产生了可见的溶解斑块。立克次体在EA.hy 926细胞中的生长与人脐静脉内皮细胞（HUVEC）中的生长相似，立克次体催化肌动蛋白尾部的聚合。立克次体胞内增殖导致EA.hy 926细胞内部形态发生显著变化，最显著的是内质网膜和外核包膜的广泛扩张（72 h p.i.），这些事件与宿主细胞抗氧化系统的显著改变有关。包括细胞内还原性谷胱甘肽和谷胱甘肽过氧化物酶活性的降低以及细胞内过氧化物含量的增加，一直持续到感染96小时。这些发现与之前描述的立克次体感染HUVEC的变化相似，表明与立克次体诱导的氧化损伤相关的共同机制发生在两种细胞系中。采用EA、hy 926细胞研究抗氧化剂硫辛酸对立克次体感染的影响。1-500 μ M α -硫辛酸隔夜预处理不能防止立克次体感染后细胞的破坏。接种立克次体2小时后，在培养基中添加1 μ M和10 μ M α -硫辛酸也没有任何保护作用。然而，100、200和500 μ M α -硫辛酸在96 h时将感染细胞的活力分别提高到45%、51%和70%，而未处理的感染样品的活力分别为26%。在处理和感染的细胞中，硫醇水平和谷胱甘肽过氧化物酶活性随着α -硫辛酸浓度的增加而增加，过氧化物含量成比例地降低。此外，500 μ M α -硫辛酸处理72 h p.i.完全阻止了感染细胞的超微结构变化。由此可见，永久内皮细胞株EA.hy 926易受立克次体感染的损伤。虽然感染引起的细胞变化在各个方面与先前在HUVEC中所证明的不同，但EA.hy 926细胞的可重复性和方便性的提高使其适合于生化和形态学研究。
EA.hy 926 is a permanent human cell line that expresses highly differentiated functions characteristic of human vascular endothelium. Rickettsia rickettsii can efficiently infect and cause a cytopathic effect in EA.hy 926 cells. R. rickettsii produced visible lytic plaques in EA.hy 926 cells at 10 d postinfection (p.i.) following application of a secondary agarose overlay containing 2 mu g emetine ml(-1) and 40 mu g NaF ml(-1) on day 2. Rickettsial growth in EA.hy 926 cells had a similar profile to that occurring in human umbilical vein endothelial cells (HUVEC) and rickettsiae catalysed polymerization of actin tails. Intracellular multiplication of R. rickettsii resulted in significant changes in the internal morphology of EA.hy 926 cells, most notably extensive dilatation of the membranes of the endoplasmic reticulum and outer nuclear envelope by 72 h p.i, These events correlated with significant alterations in the host-cell antioxidant system, including decreased levels of intracellular reduced glutathione and glutathione peroxidase activity and increased amounts of intracellular peroxide through to 96 h of infection. These findings are similar to the changes described previously for R. rickettsii-infected HUVEC and suggest that common mechanisms associated with rickettsia-induced oxidative injury occur in the two cell lines. EA,hy 926 cells were also used to investigate the influence of the antioxidant alpha-lipoic acid on rickettsial infection. Overnight pretreatment with 1-500 mu M alpha-lipoic acid did not prevent cells from being destroyed following infection with rickettsiae. Supplementation of the culture medium with 1 and 10 mu M alpha-lipoic acid 2 h after rickettsial inoculation also did not provide any protective effect. However, 100, 200 and 500 mu M alpha-lipoic acid increased the viability of infected cells at 96 h to 45, 51 and 70%, respectively compared with 26% for untreated, infected samples. Thiol levels and glutathione peroxidase activity in treated, infected cells increased and peroxide content decreased proportionally to increasing alpha-lipoic acid concentrations. Furthermore, treatment with 500 mu M alpha-lipoic acid for 72 h p.i. completely prevented ultrastructural changes in infected cells. in conclusion, the permanent endothelial cell line EA.hy 926 is susceptible to injury induced by R, rickettsii infection. Although the cellular changes resulting from infection are not identical in all aspects to that demonstrated previously in HUVEC, the increased reproducibility and convenience of EA.hy 926 cells make them suitable for biochemical and morphological studies.