呼吸道合胞病毒感染巨噬細(xì)胞誘導(dǎo)COX-2和5-LOX表達(dá)的相關(guān)研究
發(fā)布時間：2019-05-28 19:34
【摘要】： 目的: 探討呼吸道合胞病毒(respiratory syncytial virus,RSV)感染肺巨噬細(xì)胞RAW264.7細(xì)胞時,環(huán)氧化酶-2(cyclooxygenase-2,COX-2)、5-脂氧合酶(5-lipoxygenase,5-LOX)的表達(dá)及其相關(guān)炎癥介質(zhì)的水平變化,初步闡明其誘導(dǎo)表達(dá)的調(diào)控機(jī)制,為研究RSV感染時的炎癥產(chǎn)生機(jī)理,臨床治療RSV感染所致的炎癥反應(yīng)等提供一定的理論依據(jù)。 方法: ①RSV感染體外培養(yǎng)的RAW264.7巨噬細(xì)胞,分別于不同感染時間點(4h、8h、16h、24h)收集細(xì)胞和細(xì)胞培養(yǎng)上清。用半定量逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(RT-PCR)和Western-blot法分別檢測細(xì)胞中環(huán)氧化酶-2(COX-2)及5-脂氧合酶(5-LOX)的mRNA和蛋白表達(dá),酶聯(lián)免疫法(ELISA)檢測細(xì)胞培養(yǎng)上清PGE2和LTB4的含量變化。②RSV感染中加入50μmol/L NF-κB特異性抑制劑二硫代氨基吡咯烷(pyrrolidine dithiocarbamate,PDTC)抑制NF-κB的入核活化,用同樣的方法檢測上述細(xì)胞中COX-2及5-LOX的表達(dá)和細(xì)胞培養(yǎng)上清中PGE2和LTB4的變化。 結(jié)果: ①RSV感染8h后即上調(diào)RAW264.7巨噬細(xì)胞COX-2 mRNA轉(zhuǎn)錄和蛋白水平的表達(dá)。細(xì)胞培養(yǎng)上清中PGE2的含量逐漸增加。各指標(biāo)的變化與正常對照相比差異均有顯著性,并且隨著RSV感染時間的延長而表達(dá)量增加。 ②RSV感染24h內(nèi),RSW264.7巨噬細(xì)胞中5-LOX的表達(dá)及細(xì)胞培養(yǎng)上清中LTB4含量較正常對照組變化無顯著性差異。 ③RSV感染中加入PDTC后,可抑制NF-κB的入核活化,核內(nèi)NF-κBp65蛋白的表達(dá)量明顯降低;COX-2轉(zhuǎn)錄和蛋白水平的表達(dá)明顯下調(diào),在感染8h時即有明顯降低,降低具有顯著性差異。而5-LOX無論是轉(zhuǎn)錄水平還是蛋白水平的表達(dá)較對照組均無顯著性差異,同時下游炎癥因子LTB4的含量也無降低。 結(jié)論: RSV感染RAW264.7巨噬細(xì)胞早期即可誘導(dǎo)COX-2的表達(dá),同時生成大量的PGE2,PGE2的合成主要受COX-2的調(diào)節(jié),而NF-κB活化對于COX-2基因表達(dá)具有重要的調(diào)控作用;RSV感染RAW264.7巨噬細(xì)胞24h內(nèi)對5-LOX的表達(dá)無影響,同時NF-κB抑制劑對5-LOX的表達(dá)及相關(guān)炎癥因子也均無影響。
[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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R373
本文編號：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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R373
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