發(fā)布時(shí)間：2018-05-15 10:00

  本文選題：呼吸道合胞病毒 + 誘導(dǎo)型一氧化氮合酶�。� 參考：《安徽醫(yī)科大學(xué)》2008年碩士論文

【摘要】： 目的: 探討呼吸道合胞病毒(respiratory syncytial virus,RSV)感染人肺上皮細(xì)胞A549細(xì)胞時(shí),一氧化氮(nitric oxide,NO)的生成變化及其可能的調(diào)控機(jī)制,研究NO在RSV感染中的作用。 方法: ①RSV感染體外培養(yǎng)的A549細(xì)胞,分別于不同感染時(shí)間點(diǎn)(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)和免疫細(xì)胞化學(xué)法分別檢測細(xì)胞中誘導(dǎo)型一氧化氮合酶(inducible nitric oxide synthase,iNOS)mRNA和蛋白的表達(dá),免疫印跡法檢測核內(nèi)核轉(zhuǎn)錄因子κB(nuclear factor-κ-binding,NF-κB)p65蛋白的表達(dá)變化,硝酸還原酶法和硫代巴比妥酸法分別檢測細(xì)胞培養(yǎng)上清NO和自由基氧化損傷產(chǎn)物指標(biāo)丙二醛(malondialdehyde,MDA)的含量,化學(xué)法檢測細(xì)胞培養(yǎng)上清中活性氧指標(biāo)羥自由基(hydroxy radical,OH·)與超氧陰離子(superoxide anion,O2.￣)的水平,空斑形成實(shí)驗(yàn)檢測細(xì)胞培養(yǎng)上清中病毒復(fù)制滴度指標(biāo)空斑形成單位數(shù)(plaque forming unit,PFU);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iNOS的表達(dá);③RSV感染中加入1mmol/L iNOS特異性抑制劑氨基胍(Aminoguanidine,AG)抑制NO的合成,分別檢測細(xì)胞培養(yǎng)上清中NO、OH·、O2.￣、MDA和PFU的變化。 結(jié)果: ①RSV感染4 h后即上調(diào)A549細(xì)胞iNOS mRNA轉(zhuǎn)錄和蛋白水平的表達(dá)。4h后即可誘導(dǎo)核內(nèi)NF-κBp65蛋白的表達(dá)量升高,8h后具有明顯升高。細(xì)胞培養(yǎng)上清中NO、OH·和O2.￣的含量逐漸增加,MDA水平顯著增加,病毒復(fù)制滴度PFU升高。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(shí)間的延長而表達(dá)量增加。 ②RSV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;iNOS mRNA轉(zhuǎn)錄和蛋白水平的表達(dá)明顯下調(diào),在感染4h時(shí)即有明顯降低,降低具有顯著性差異。 ③RSV感染中加入AG后,可抑制iNOS合成NO;降低細(xì)胞培養(yǎng)上清中OH·和O2.￣的含量,O2.￣在感染16h后有顯著性降低,OH·在感染24h后有顯著性降低,MDA含量均有明顯下降。但細(xì)胞培養(yǎng)上清中的病毒PFU均升高。 結(jié)論: RSV感染A549細(xì)胞可誘導(dǎo)生成大量的NO,NO的合成主要受iNOS的調(diào)節(jié)。NF-κB活化對于iNOS基因表達(dá)具有重要的調(diào)控作用。NO可引起細(xì)胞內(nèi)自由基水平升高,加重細(xì)胞的自由基損傷程度,但可降低病毒在感染中的復(fù)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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R373

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