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目的探讨腺病毒早表达蛋白1A(E1A)对脂多糖、肿瘤坏死因子α(TNF-α)诱导的大鼠肺泡上皮细胞炎症介质细胞间黏附分子1(ICAM-1)表达的影响及其机制。方法致炎因素脂多糖和 TNF-α作用于稳定表达 E1A 的大鼠肺泡卜皮细胞、对照质粒转染细胞和正常大鼠肺泡上皮细胞,采用流式单抗和 RT-PCR 法分析 ICAM-1蛋白水平和 mRNA 水平的表达情况;转录因子报告系统和凝胶电泳迁移变动分析(EMSA)研究核因子(NF-κB)、活化蛋白1与 ICAM-1基因上游调控元件结合的情况。结果 (1)与对照质粒组和正常细胞组相比,E1A 阳性组细胞经10 mg/L 脂多糖和10 pg/L TNF-α刺激后,ICAM-1蛋白表达(任意荧光强度)为109±15和185±20,比对照质粒转染组细胞(60±13,86±22)和正常 CCL149细胞(61±20,89±12)明显升高(F 值分别为14.46、73.64,P 均<0.01);(2)RT-PCR 显示 E1A 阳性组细胞 ICAM-1 mRNA 的表达在脂多糖、TNF-α刺激后3h和6h 均比对照质粒转染组细胞明显增高;(3)转录因子荧光素酶报道系统及 EMSA 结果显示,E1A组在脂多糖、TNF-α作用前后,细胞核内 NF-κB与 ICAM-1基因上游调控序列结合形成的阻滞条带强度均明显高于埘照组;而活化蛋白1与 ICAM-1基因上游调控序列特异性结合形成的阻滞条带在E1A 阳性组和对照组在刺激因素作用前后比较无明显差异;(4)在脂多糖作用后,NF-κB抑制剂 N-甲苯磺基-L-苯乙胺酰氯甲基酮(TPCK)可使 E1A 组 ICAM-1蛋白表达强度下降(109±15,50±10);TNF-α作用后 E1A 组 ICAM-1蛋白表达强度也明显下降(185±20,55±13),TPCK 处理前后比较,差异有统计学意义(t 值分别为8.4、12.2,P 值分别为0.01和0.00)。结论 E1A 可明显上调炎症介质ICAM-1的表达,上调转录因子 NF-κB、活化蛋白1的活性;E1A 上调 ICAM-1的表达主要通过 NF-κB实现。
Objective To investigate the effect of adenovirus E1A on the expression of ICAM-1 in rat alveolar epithelial cells induced by lipopolysaccharide and tumor necrosis factor α (TNF-α). Methods The inflammatory factors lipopolysaccharide and TNF-α were used to effect the lung alveolar epithelial cells stably expressing E1A, the control plasmid transfected cells and the normal rat alveolar epithelial cells. Flow cytometry and RT-PCR were used to analyze ICAM-1 Protein level and mRNA level. The transcription factor reporter system and electrophoretic mobility shift assay (EMSA) were used to study the binding of nuclear factor (NF-κB) and activin-1 to upstream regulatory elements of ICAM-1 gene. Results (1) Compared with the control plasmid group and the normal cell group, the expression of ICAM-1 protein (arbitrary fluorescence intensity) in E1A positive cells stimulated by 10 mg / L lipopolysaccharide and 10 pg / L TNF-α was 109 ± 15 and 185 ± 20 were significantly higher than those of the control plasmid transfected cells (60 ± 13,86 ± 22) and normal CCL149 cells (61 ± 20,89 ± 12) (F = 14.46 and 73.64, P < 0.01). (2) The expression of ICAM-1 mRNA in E1A positive group was significantly higher than that in control plasmid transfected group at 3h and 6h after stimulation with lipopolysaccharide and TNF-α by RT-PCR. (3) The transcription factor fluorescein The results of EMSA and EMSA showed that the intensity of blocking bands in NF-κB and ICAM-1 upstream regulatory sequences in E1A group were significantly higher than those in control group before and after lipopolysaccharide and TNF-α treatment There was no significant difference between the E1A positive group and the control group before and after the stimulation of the activating protein 1 and ICAM-1 gene upstream regulatory sequences; (4) NF-κB inhibition The intensity of ICAM-1 protein expression in E1A group was decreased (109 ± 15,50 ± 10) by agent N-tolylsulfo-L-phenylethylamine chloride methyl ketone (TPCK) The expression of ICAM-1 protein was also significantly decreased (185 ± 20,55 ± 13). There was significant difference between before and after TPCK treatment (t = 8.4 and 12.2, P = 0.01 and 0.00, respectively). Conclusion E1A can up-regulate the expression of ICAM-1 in inflammatory mediators and up-regulate the activity of transcription factors NF-κB and activator protein 1. E1A up-regulates the expression of ICAM-1 through NF-κB.