一氧化氮在呼吸道合胞病毒感染人肺上皮细胞中的生成机制及其作用

发布时间：2018-05-15 10:00

本文选题：呼吸道合胞病毒 + 诱导型一氧化氮合酶　；参考：《安徽医科大学》2008年硕士论文

【摘要】： 目的: 探讨呼吸道合胞病毒(respiratory syncytial virus,RSV)感染人肺上皮细胞A549细胞时,一氧化氮(nitric oxide,NO)的生成变化及其可能的调控机制,研究NO在RSV感染中的作用。方法: ①RSV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)和免疫细胞化学法分别检测细胞中诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)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,硝酸还原酶法和硫代巴比妥酸法分别检测细胞培养上清NO和自由基氧化损伤产物指标丙二醛(malondialdehyde,MDA)的含量,化学法检测细胞培养上清中活性氧指标羟自由基(hydroxy radical,OH·)与超氧阴离子(superoxide anion,O2.￣)的水平,空斑形成实验检测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);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iNOS的表达;③RSV感染中加入1mmol/L iNOS特异性抑制剂氨基胍(Aminoguanidine,AG)抑制NO的合成,分别检测细胞培养上清中NO、OH·、O2.￣、MDA和PFU的变化。结果: ①RSV感染4 h后即上调A549细胞iNOS mRNA转录和蛋白水平的表达。4h后即可诱导核内NF-κBp65蛋白的表达量升高,8h后具有明显升高。细胞培养上清中NO、OH·和O2.￣的含量逐渐增加,MDA水平显著增加,病毒复制滴度PFU升高。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;iNOS mRNA转录和蛋白水平的表达明显下调,在感染4h时即有明显降低,降低具有显著性差异。 ③RSV感染中加入AG后,可抑制iNOS合成NO;降低细胞培养上清中OH·和O2.￣的含量,O2.￣在感染16h后有显著性降低,OH·在感染24h后有显著性降低,MDA含量均有明显下降。但细胞培养上清中的病毒PFU均升高。结论: RSV感染A549细胞可诱导生成大量的NO,NO的合成主要受iNOS的调节。NF-κB活化对于iNOS基因表达具有重要的调控作用。NO可引起细胞内自由基水平升高,加重细胞的自由基损伤程度,但可降低病毒在感染中的复制滴度。
[Abstract]:Objective: To investigate the changes of nitric oxide (no) production in human pulmonary epithelial cells (A549) infected with respiratory syncytial virus (RSVV) and its possible regulatory mechanism, and to investigate the role of no in RSV infection. Methods: A549 cells infected with 1RSV were collected and supernatant of cell culture were collected at different infection time points (4 h, 8 h, 16 h, 24 h). Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry were used to detect the expression of inducible nitric oxide synthase (inducible nitric oxide synthase) mRNA and protein, and the expression of nuclear transcription factor 魏 B(nuclear factor- 魏 -binding NF- 魏 B)p65 were detected by Western blot. Nitric acid reductase method and thiobarbituric acid method were used to detect the content of no in cell culture supernatant and malondialdehyde (MDA) in cell culture supernatant. The levels of hydroxyl radical OH and superoxide anion superoxide radical O _ 2. (2) in superoxide supernatant were detected by chemical method, and the levels of hydroxyl radical (OH) and superoxide anion (superoxide anion) were measured by chemical method. Plaque formation assay to detect the titer of virus replication in the supernatant of cell culture, plaque forming unit number of plaque forming units and the addition of 50 渭 mol/L NF- 魏 B specific inhibitor pyrrolidine dithiocarbamatea PDTCto inhibit the activation of NF- 魏 B into the nucleus of the supernatant, and the inhibition of the activation of NF- 魏 B by the addition of 50 渭 mol/L NF- 魏 B specific inhibitor, pyrrolidine dithiocarbamatea (PDTCc), was observed. The expression of iNOS in the above mentioned cells was detected by the same method. Aminoguanidine (AGG), a specific inhibitor of Aminoguanidine, was added to the above cells to inhibit the synthesis of no, and the changes of no OH O 2 and PFU in the supernatant of cell culture were detected respectively. Results: After 4 h of 1RSV infection, the expression of iNOS mRNA transcription and protein in A549 cells was upregulated. 4 h after infection, the expression of NF- 魏 Bp65 in A549 cells could be induced to increase significantly after 8 h. In the supernatant of cell culture, the content of NOOOH and O2.The content of PFU in the supernatant increased significantly, and the replication titer of PFU increased significantly. The changes of each index were significantly different from those of normal control, and the expression level increased with the prolongation of RSV infection time. The activation of NF- 魏 B was inhibited by the addition of PDTC to 2RSV infection, and the expression of NF- 魏 Bp65 protein decreased significantly. The expression of NF- 魏 Bp65 mRNA decreased significantly at 4 h after infection, and there was significant difference in the decrease of NF- 魏 B expression. After adding AG to 3RSV infection, iNOS synthesis was inhibited, and the contents of OH and O _ 2 路O _ 2 in supernatant of cell culture were decreased significantly. But the virus PFU in the supernatant of cell culture was increased. Conclusion: RSV infected A549 cells could induce the synthesis of a large amount of NON- no, which was mainly regulated by iNOS. NF- 魏 B activation had an important regulatory effect on the expression of iNOS gene. No could increase the level of intracellular free radicals and aggravate the degree of free radical damage in A549 cells. But it can reduce the replication titer of virus in infection.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R373

【相似文献】