【摘要】： 目的: 探討呼吸道合胞病毒(respiratory syncytial virus,RSV)感染A549細(xì)胞及BALB/c鼠COX-2表達(dá)的變化及其可能的調(diào)控機(jī)制,初步研究COX-2在RSV感染所致炎性反應(yīng)中的作用。 方法: ①1×10~6 PFU/ml RSV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 )、免疫細(xì)胞化學(xué)法和免疫印跡法分別檢測細(xì)胞中COX-2(cyclooxygenase 2,COX-2)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;用酶聯(lián)免疫法(ELISA)檢測細(xì)胞培養(yǎng)上清的PGE2表達(dá)變化;空斑形成實(shí)驗(yàn)測定細(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)。1×106PFU/ml RSV滴鼻感染BALB/c鼠,在感染后24h、48h和96h,用免疫組化法和免疫印跡法檢測肺組織COX-2蛋白表達(dá),并對(duì)肺組織切片行HE染色,觀察肺組織炎癥病理變化。以未感染病毒的A549細(xì)胞及BALB/c鼠作正常對(duì)照,采用生物圖像分析系統(tǒng)對(duì)免疫組織化學(xué)和免疫細(xì)胞化學(xué)結(jié)果進(jìn)行COX-2定量檢測。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,用半定量RT-PCR法、免疫細(xì)胞化學(xué)法和免疫印跡法分別檢測A549細(xì)胞中COX-2的表達(dá);③RSV感染中加入100μmol/L COX-2特異性抑制劑尼美舒利抑制PGE2的合成,分別檢測細(xì)胞培養(yǎng)上清中PGE2和病毒復(fù)制滴度的變化。 結(jié)果: ①正常細(xì)胞對(duì)照組中,COX-2 mRNA和蛋白有基礎(chǔ)表達(dá),經(jīng)由RSV感染后,A549細(xì)胞COX-2 mRNA和蛋白有高水平表達(dá),差異具有統(tǒng)計(jì)學(xué)意義(P0.05),且隨著感染時(shí)間的延長,其表達(dá)量逐漸增加。正常鼠肺組織中COX-2 mRNA和蛋白僅有微弱表達(dá),RSV感染后,COX-2 mRNA和蛋白表達(dá)水平隨著感染時(shí)間延長而增加,差異具有統(tǒng)計(jì)學(xué)意義(P0.05);HE染色顯示肺組織炎癥程度隨感染時(shí)間延長而加重,且COX-2的表達(dá)水平與肺組織炎癥嚴(yán)重程度相一致。RSV感染4h后即可誘導(dǎo)A549細(xì)胞核內(nèi)NF-κB p65蛋白的表達(dá)量升高,8h后具有顯著升高(P0.01)。細(xì)胞培養(yǎng)上清中PGE2的含量逐漸增加,病毒復(fù)制滴度PFU升高,隨著RSV感染時(shí)間的延長而含量增加,與正常對(duì)照組相比差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。 ②RSV感染中加入PDTC后,可抑制A549細(xì)胞NF-κB的入核活化,核內(nèi)NF-κB p65蛋白的表達(dá)量顯著降低;COX-2 mRNA轉(zhuǎn)錄水平和蛋白水平的表達(dá)顯著下調(diào),在感染8h時(shí)即有降低,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。 ③RSV感染中加入尼美舒利后,可抑制COX-2合成PGE2;降低細(xì)胞培養(yǎng)上清中PGE2的含量,差異具有統(tǒng)計(jì)學(xué)(P0.05);但細(xì)胞培養(yǎng)上清中的病毒PFU變化無統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論: RSV感染可誘導(dǎo)A549細(xì)胞及肺組織高水平表達(dá)COX-2 mRNA和蛋白,且PGE2的含量隨著RSV感染時(shí)間的延長而增加;并可能參與了RSV感染引起的肺組織炎性反應(yīng)。NF-κB活化對(duì)于COX-2基因表達(dá)具有重要的正調(diào)控作用,PGE2的合成受到COX-2的調(diào)控,而病毒復(fù)制水平未受COX-2活性水平的影響。
[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.
【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】：2009
【分類號(hào)】：R373

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本文編號(hào)：2234974