呼吸道合胞病毒感染巨噬细胞诱导COX-2和5-LOX表达的相关研究
发布时间：2019-05-28 19:34
【摘要】： 目的: 探讨呼吸道合胞病毒(respiratory syncytial virus,RSV)感染肺巨噬细胞RAW264.7细胞时,环氧化酶-2(cyclooxygenase-2,COX-2)、5-脂氧合酶(5-lipoxygenase,5-LOX)的表达及其相关炎症介质的水平变化,初步阐明其诱导表达的调控机制,为研究RSV感染时的炎症产生机理,临床治疗RSV感染所致的炎症反应等提供一定的理论依据。 方法: ①RSV感染体外培养的RAW264.7巨噬细胞,分别于不同感染时间点(4h、8h、16h、24h)收集细胞和细胞培养上清。用半定量逆转录聚合酶链反应(RT-PCR)和Western-blot法分别检测细胞中环氧化酶-2(COX-2)及5-脂氧合酶(5-LOX)的mRNA和蛋白表达,酶联免疫法(ELISA)检测细胞培养上清PGE2和LTB4的含量变化。②RSV感染中加入50μmol/L NF-κB特异性抑制剂二硫代氨基吡咯烷(pyrrolidine dithiocarbamate,PDTC)抑制NF-κB的入核活化,用同样的方法检测上述细胞中COX-2及5-LOX的表达和细胞培养上清中PGE2和LTB4的变化。 结果: ①RSV感染8h后即上调RAW264.7巨噬细胞COX-2 mRNA转录和蛋白水平的表达。细胞培养上清中PGE2的含量逐渐增加。各指标的变化与正常对照相比差异均有显著性,并且随着RSV感染时间的延长而表达量增加。 ②RSV感染24h内,RSW264.7巨噬细胞中5-LOX的表达及细胞培养上清中LTB4含量较正常对照组变化无显著性差异。 ③RSV感染中加入PDTC后,可抑制NF-κB的入核活化,核内NF-κBp65蛋白的表达量明显降低;COX-2转录和蛋白水平的表达明显下调,在感染8h时即有明显降低,降低具有显著性差异。而5-LOX无论是转录水平还是蛋白水平的表达较对照组均无显著性差异,同时下游炎症因子LTB4的含量也无降低。 结论: RSV感染RAW264.7巨噬细胞早期即可诱导COX-2的表达,同时生成大量的PGE2,PGE2的合成主要受COX-2的调节,而NF-κB活化对于COX-2基因表达具有重要的调控作用;RSV感染RAW264.7巨噬细胞24h内对5-LOX的表达无影响,同时NF-κB抑制剂对5-LOX的表达及相关炎症因子也均无影响。
[Abstract]:Objective: to investigate the infection of pulmonary macrophage RAW264.7 cells with respiratory syncytial virus (respiratory syncytial virus,RSV). Cyclooxygenase-2 (cyclooxygenase-2,COX-2) and 5-lipoxygenase (5-lipoxygenase) were used to investigate the infection of pulmonary macrophages with Cox-2 and 5-lipoxygenase. The expression of 5-LOX) and the changes of its related inflammatory mediators were preliminarily clarified, which provided a theoretical basis for the study of the mechanism of inflammation caused by RSV infection and the clinical treatment of inflammatory response caused by RSV infection. Methods: RAW264.7 macrophages infected with 1RSV in vitro were collected at different infection time points (4 h, 8 h, 16 h, 24 h). The mRNA and protein expressions of cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) in cells were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western-blot, respectively. Enzyme-linked immunosorbent assay (Elisa) (ELISA) was used to detect the contents of PGE2 and LTB4 in cell culture. 2the addition of 50 渭 mol / L NF- 魏 B specific inhibitor dithioaminopyrrolidine (pyrrolidine dithiocarbamate,PDTC) to RSV infection inhibited the nucleation and activation of NF- 魏 B. The expression of COX-2 and 5-LOX in the above cells and the changes of PGE2 and LTB4 in the cell culture supernatant were detected by the same method. Results: the expression of COX-2 mRNA transcription and protein in RAW264.7 macrophages was up-regulated 8 hours after 1RSV infection. The content of PGE2 in the cell culture medium increased gradually. The changes of each index were significantly different from those of the normal control, and the expression increased with the prolongation of RSV infection time. Within 24 hours of 2RSV infection, there was no significant difference in the expression of 5-LOX in RSW264.7 macrophages and the content of LTB4 in cell culture supernatant compared with the normal control group. The addition of PDTC to 3RSV infection could inhibit the nucleation and activation of NF- 魏 B and decrease the expression of NF- 魏 Bp65 protein in the nucleus. The expression of COX-2 transcription and protein level was significantly down-regulated, which decreased significantly at 8 h after infection, and there was significant difference between the two groups. However, the expression of 5-LOX at transcriptional level and protein level was not significantly different from that in the control group, and the content of downstream inflammatory factor LTB4 did not decrease. Conclusion: RSV infection can induce the expression of COX-2 in RAW264.7 macrophages at an early stage, and the synthesis of a large number of PGE2,PGE2 is mainly regulated by COX-2. The activation of NF- kappa B plays an important role in the regulation of COX-2 gene expression. RAW264.7 macrophages infected with RSV had no effect on the expression of 5-LOX within 24 hours, and NF- kappa B inhibitor had no effect on the expression of 5-LOX and related inflammatory factors.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R373
本文编号：2487313
[Abstract]:Objective: to investigate the infection of pulmonary macrophage RAW264.7 cells with respiratory syncytial virus (respiratory syncytial virus,RSV). Cyclooxygenase-2 (cyclooxygenase-2,COX-2) and 5-lipoxygenase (5-lipoxygenase) were used to investigate the infection of pulmonary macrophages with Cox-2 and 5-lipoxygenase. The expression of 5-LOX) and the changes of its related inflammatory mediators were preliminarily clarified, which provided a theoretical basis for the study of the mechanism of inflammation caused by RSV infection and the clinical treatment of inflammatory response caused by RSV infection. Methods: RAW264.7 macrophages infected with 1RSV in vitro were collected at different infection time points (4 h, 8 h, 16 h, 24 h). The mRNA and protein expressions of cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) in cells were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western-blot, respectively. Enzyme-linked immunosorbent assay (Elisa) (ELISA) was used to detect the contents of PGE2 and LTB4 in cell culture. 2the addition of 50 渭 mol / L NF- 魏 B specific inhibitor dithioaminopyrrolidine (pyrrolidine dithiocarbamate,PDTC) to RSV infection inhibited the nucleation and activation of NF- 魏 B. The expression of COX-2 and 5-LOX in the above cells and the changes of PGE2 and LTB4 in the cell culture supernatant were detected by the same method. Results: the expression of COX-2 mRNA transcription and protein in RAW264.7 macrophages was up-regulated 8 hours after 1RSV infection. The content of PGE2 in the cell culture medium increased gradually. The changes of each index were significantly different from those of the normal control, and the expression increased with the prolongation of RSV infection time. Within 24 hours of 2RSV infection, there was no significant difference in the expression of 5-LOX in RSW264.7 macrophages and the content of LTB4 in cell culture supernatant compared with the normal control group. The addition of PDTC to 3RSV infection could inhibit the nucleation and activation of NF- 魏 B and decrease the expression of NF- 魏 Bp65 protein in the nucleus. The expression of COX-2 transcription and protein level was significantly down-regulated, which decreased significantly at 8 h after infection, and there was significant difference between the two groups. However, the expression of 5-LOX at transcriptional level and protein level was not significantly different from that in the control group, and the content of downstream inflammatory factor LTB4 did not decrease. Conclusion: RSV infection can induce the expression of COX-2 in RAW264.7 macrophages at an early stage, and the synthesis of a large number of PGE2,PGE2 is mainly regulated by COX-2. The activation of NF- kappa B plays an important role in the regulation of COX-2 gene expression. RAW264.7 macrophages infected with RSV had no effect on the expression of 5-LOX within 24 hours, and NF- kappa B inhibitor had no effect on the expression of 5-LOX and related inflammatory factors.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R373
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