呼吸道合胞病毒诱导COX-2表达及其部分机制的研究
发布时间：2018-09-10 16:21
【摘要】： 目的: 探讨呼吸道合胞病毒(respiratory syncytial virus,RSV)感染A549细胞及BALB/c鼠COX-2表达的变化及其可能的调控机制,初步研究COX-2在RSV感染所致炎性反应中的作用。 方法: ①1×10~6 PFU/ml RSV感染体外培养的A549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 )、免疫细胞化学法和免疫印迹法分别检测细胞中COX-2(cyclooxygenase 2,COX-2)mRNA和蛋白的表达;免疫印迹法检测核内核转录因子κB(nuclear factor-κ-binding,NF-κB)p65蛋白的表达变化;用酶联免疫法(ELISA)检测细胞培养上清的PGE2表达变化;空斑形成实验测定细胞培养上清中病毒复制滴度指标空斑形成单位数(plaque forming unit,PFU)。1×106PFU/ml RSV滴鼻感染BALB/c鼠,在感染后24h、48h和96h,用免疫组化法和免疫印迹法检测肺组织COX-2蛋白表达,并对肺组织切片行HE染色,观察肺组织炎症病理变化。以未感染病毒的A549细胞及BALB/c鼠作正常对照,采用生物图像分析系统对免疫组织化学和免疫细胞化学结果进行COX-2定量检测。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,用半定量RT-PCR法、免疫细胞化学法和免疫印迹法分别检测A549细胞中COX-2的表达;③RSV感染中加入100μmol/L COX-2特异性抑制剂尼美舒利抑制PGE2的合成,分别检测细胞培养上清中PGE2和病毒复制滴度的变化。 结果: ①正常细胞对照组中,COX-2 mRNA和蛋白有基础表达,经由RSV感染后,A549细胞COX-2 mRNA和蛋白有高水平表达,差异具有统计学意义(P0.05),且随着感染时间的延长,其表达量逐渐增加。正常鼠肺组织中COX-2 mRNA和蛋白仅有微弱表达,RSV感染后,COX-2 mRNA和蛋白表达水平随着感染时间延长而增加,差异具有统计学意义(P0.05);HE染色显示肺组织炎症程度随感染时间延长而加重,且COX-2的表达水平与肺组织炎症严重程度相一致。RSV感染4h后即可诱导A549细胞核内NF-κB p65蛋白的表达量升高,8h后具有显著升高(P0.01)。细胞培养上清中PGE2的含量逐渐增加,病毒复制滴度PFU升高,随着RSV感染时间的延长而含量增加,与正常对照组相比差异具有统计学意义(P0.05)。 ②RSV感染中加入PDTC后,可抑制A549细胞NF-κB的入核活化,核内NF-κB p65蛋白的表达量显著降低;COX-2 mRNA转录水平和蛋白水平的表达显著下调,在感染8h时即有降低,差异具有统计学意义(P0.05)。 ③RSV感染中加入尼美舒利后,可抑制COX-2合成PGE2;降低细胞培养上清中PGE2的含量,差异具有统计学(P0.05);但细胞培养上清中的病毒PFU变化无统计学意义(P0.05)。 结论: RSV感染可诱导A549细胞及肺组织高水平表达COX-2 mRNA和蛋白,且PGE2的含量随着RSV感染时间的延长而增加;并可能参与了RSV感染引起的肺组织炎性反应。NF-κB活化对于COX-2基因表达具有重要的正调控作用,PGE2的合成受到COX-2的调控,而病毒复制水平未受COX-2活性水平的影响。
[Abstract]:Objective:
Objective To investigate the expression of COX-2 in A549 cells and BALB/c mice infected with respiratory syncytial virus (RSV) and its possible regulatory mechanism, and to investigate the role of COX-2 in the inflammatory response induced by RSV infection.
Method:
The supernatants of A549 cells were collected at different time points (4h, 8h, 16h, 24h) and the expression of COX-2 (COX-2) mRNA and protein were detected by Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), immunocytochemistry and Western blot respectively. The expression of nuclear factor-kappa-binding (NF-kappa B) p65 protein was detected by Western blot, the expression of PGE2 in cell culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA), and the plaque forming unit (PFU) was determined by plaque forming assay. The expression of COX-2 protein in lung tissues was detected by immunohistochemistry and Western blot at 24, 48 and 96 hours after RSV nasal Dropping infection in BALB/c mice. The pathological changes of lung tissue inflammation were observed by HE staining. The A549 cells and BALB/c mice were used as normal controls and the immunohistochemistry was performed by using Bio-image analysis system. COX-2 expression in A549 cells was detected by semi-quantitative RT-PCR, immunocytochemistry and Western blotting respectively. The synthesis of PGE 2 was inhibited by nimesulide, a 100 micromol/L COX-2 specific inhibitor, in V infection. The changes of PGE 2 and virus replication titer in cell culture supernatant were detected.
Result:
The expression of COX-2 mRNA and protein in A549 cells was significantly higher than that in normal control group (P 0.05). The expression of COX-2 mRNA and protein in normal lung tissues was only slightly increased with the prolongation of infection time. After RSV infection, COX-2 mRNA and protein in normal lung tissues were only slightly expressed. The expression of NF-kappa B p65 protein in the nucleus of A549 cells increased 4 hours after RSV infection. The content of PGE2 in cell culture supernatant increased gradually, the viral replication titer PFU increased, and the content increased with the extension of RSV infection time. The difference was statistically significant compared with the normal control group (P 0.05).
(2) PDTC could inhibit the activation of NF-kappa B and decrease the expression of NF-kappa B p65 protein in A549 cells, and COX-2 mRNA transcription and protein expression were down-regulated significantly after RSV infection. The expression of COX-2 mRNA was down-regulated at 8 h after RSV infection, and the difference was statistically significant (P 0.05).
(3) Nimesulide could inhibit the synthesis of PGE2 in COX-2 and decrease the content of PGE2 in cell culture supernatant, but there was no significant difference in the change of PFU in cell culture supernatant (P 0.05).
Conclusion:
RSV infection can induce the high level expression of COX-2 mRNA and protein in A549 cells and lung tissues, and the content of PGE 2 increases with the prolongation of RSV infection, and may participate in the inflammatory reaction of lung tissues induced by RSV infection.NF-kappa B activation has an important positive regulatory role in the expression of COX-2 gene, PGE 2 synthesis is regulated by COX-2, while the virus Replication level was not affected by COX-2 activity level.
【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：2234974
[Abstract]:Objective:
Objective To investigate the expression of COX-2 in A549 cells and BALB/c mice infected with respiratory syncytial virus (RSV) and its possible regulatory mechanism, and to investigate the role of COX-2 in the inflammatory response induced by RSV infection.
Method:
The supernatants of A549 cells were collected at different time points (4h, 8h, 16h, 24h) and the expression of COX-2 (COX-2) mRNA and protein were detected by Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), immunocytochemistry and Western blot respectively. The expression of nuclear factor-kappa-binding (NF-kappa B) p65 protein was detected by Western blot, the expression of PGE2 in cell culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA), and the plaque forming unit (PFU) was determined by plaque forming assay. The expression of COX-2 protein in lung tissues was detected by immunohistochemistry and Western blot at 24, 48 and 96 hours after RSV nasal Dropping infection in BALB/c mice. The pathological changes of lung tissue inflammation were observed by HE staining. The A549 cells and BALB/c mice were used as normal controls and the immunohistochemistry was performed by using Bio-image analysis system. COX-2 expression in A549 cells was detected by semi-quantitative RT-PCR, immunocytochemistry and Western blotting respectively. The synthesis of PGE 2 was inhibited by nimesulide, a 100 micromol/L COX-2 specific inhibitor, in V infection. The changes of PGE 2 and virus replication titer in cell culture supernatant were detected.
Result:
The expression of COX-2 mRNA and protein in A549 cells was significantly higher than that in normal control group (P 0.05). The expression of COX-2 mRNA and protein in normal lung tissues was only slightly increased with the prolongation of infection time. After RSV infection, COX-2 mRNA and protein in normal lung tissues were only slightly expressed. The expression of NF-kappa B p65 protein in the nucleus of A549 cells increased 4 hours after RSV infection. The content of PGE2 in cell culture supernatant increased gradually, the viral replication titer PFU increased, and the content increased with the extension of RSV infection time. The difference was statistically significant compared with the normal control group (P 0.05).
(2) PDTC could inhibit the activation of NF-kappa B and decrease the expression of NF-kappa B p65 protein in A549 cells, and COX-2 mRNA transcription and protein expression were down-regulated significantly after RSV infection. The expression of COX-2 mRNA was down-regulated at 8 h after RSV infection, and the difference was statistically significant (P 0.05).
(3) Nimesulide could inhibit the synthesis of PGE2 in COX-2 and decrease the content of PGE2 in cell culture supernatant, but there was no significant difference in the change of PFU in cell culture supernatant (P 0.05).
Conclusion:
RSV infection can induce the high level expression of COX-2 mRNA and protein in A549 cells and lung tissues, and the content of PGE 2 increases with the prolongation of RSV infection, and may participate in the inflammatory reaction of lung tissues induced by RSV infection.NF-kappa B activation has an important positive regulatory role in the expression of COX-2 gene, PGE 2 synthesis is regulated by COX-2, while the virus Replication level was not affected by COX-2 activity level.
【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
【参考文献】
相关期刊论文 前2条
1 云云;黄升海;;Toll样受体3介导呼吸道合胞病毒感染人肺上皮细胞的炎性反应及其信号转导通路[J];中国组织工程研究与临床康复;2007年21期
2 刘伟;云云;黄升海;;人肺上皮细胞感染呼吸道合胞病毒后核内活性因子κB与诱导型一氧化氮合酶的表达[J];中国组织工程研究与临床康复;2008年15期
，本文编号：2234974
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2234974.html
最近更新
教材专著
