E1A激活基因阻遏子对抗肿瘤坏死因子α引起的血管内皮细胞炎性损伤

Protection of Cellular Repressor of E1A Stimulated Genes Against TNF-α-Mediated Inflammatory Injury of Vascular Endothelial Cells

目的探讨E1A激活基因阻遏子表达对血管内皮细胞病理性炎症反应的生物学作用及机制。方法以E1A激活基因阻遏子过表达和基因沉默的人动脉血管内皮细胞为细胞模型,应用肿瘤坏死因子α刺激,ELISA检测白细胞介素6的变化,Rhodamin-Phalloidin检测肌动蛋白骨架F-actin分布,应用Transwell小室和Biotin标记的BSA检测单层内皮细胞通透性改变。进一步通过免疫荧光染色和Western blot检测细胞内核因子κB蛋白表达和转位情况。结果 ELISA检测发现,E1A激活基因阻遏子基因过表达显著抑制肿瘤坏死因子α刺激引起血管内皮细胞白细胞介素6的表达及分泌增加。同时,细胞F-actin应力纤维的形成和细胞单层通透性增加受到明显抑制。相反,E1A激活基因阻遏子基因沉默组白细胞介素6分泌较对照组明显增多。细胞F-actin应力纤维形成、细胞单层通透性显著增加。免疫荧光和Western blot分析证实,肿瘤坏死因子α刺激后,E1A激活基因阻遏子过表达组核因子κB出现短时的入核现象并迅速出核,对应蛋白出现相应的表达变化,而E1A激活基因阻遏子基因沉默组核因子κB表达持续增高且发生明显的入核转位现象。结论 E1A激活基因阻遏子可通过减少核因子κB的表达对抗肿瘤坏死因子α刺激引起的血管内皮细胞炎症反应和细胞通透性增加,对抗细胞病理性损伤发生。

Aim To clarify the pathological effects and mechanisms of cellular repressor of E1A stimulated gene （CREG） on tumor necrosis factor-or （TNF-α） stimulated vascular endothelial cell （VEC） injury. Methods CREG overexpressed （VC） and knocked-down （VS） human artery VEC were produced and cells were exposed to TNF-α （ 10 Ixg/ L）. Interleukin-6 （IL-6） secretion from cells was determined by enzyme immunolinked assay （ELISA）. Filamentous actin （F-actin） stress fibre were detected by Rhodamin-Phalloidin staining. Endothelial permeability was detected by measuring the flux of biotin labeled albumin across the EC monolayers. Nuclear factor-KB （NF-KB） expressions and translocalization were examined by Western blot analysis and immunofluorescence. Results After TNF-α stimulation, ELISA showed that IL-6 expression and secretion in VS cells was markedly increased as well as F-actin cytoskeleton rearrangement. Meanwhile, the permeability of VS cells was detected enhanced obviously. Conversely, overexpression of CREG inhibited the secretion of IL-6, F-actin stress fibre formation and hyperpermeability induced by TNF-α in VC cells.Moreover, immunoflurosence and Western blot showed that NF-KB transiently translocated into the nuclei of VC cells, foliowed by quick exportation into the cytoplasm. Corresponding changes in the pattern of its expression was also observed. However, the expression of NF-KB in CREG knocked-down VS cells was more sustainably elevated and retained in the nuclei. Conclusions CREG can inhibit NF-KB expression, combat TNF-α-induced inflammatory responses and the hyperpermeability of VEC, and thereby antagonize pathological cellular injury.