H7N9禽流感病毒感染A549细胞生物标志物研究
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摘要
目的通过建立H7N9禽流感病毒和H1N1pdm09流感病毒(对照病毒)感染A549细胞模型,分析H7N9禽流感病毒感染A549细胞的特征蛋白,筛选其生物标志物,初步揭示H7N9禽流感病毒致细胞病变的蛋白分子机制。方法感染复数为0.001的H7N9和对照病毒分别感染A549细胞24、48、72 h后提取细胞总蛋白进行荧光双向差异凝胶电泳(2D-DIGE),筛选H7N9禽流感病毒感染A549细胞的可能生物标志物并进行蛋白印迹(WB)验证。结果血小板活化因子乙酰水解酶Ⅰb亚基β(PAFAH1B2)蛋白在H7N9感染A549细胞72 h后表达量急剧下降,而在空白对照组和H1N1pdm09感染A549细胞组的表达量未出现明显变化。结论 PAFAH1B2可作为H7N9感染A549细胞后期的生物标志物,其表达量的变化可能与H7N9感染患者的症状相关。
Objective To analyze specific protein and screen biomarkers of H7N9 avian influenza virus infection in A549 cells by establishing a A549 cell model of H7N9 avian influenza virus and H1N1 pdm09 influenza virus(the control)infection for exploring the pathogenic mechanism of H7N9 avian influenza virus infection.Methods The A549 cells were infected by H7N9 avian influenza virus with the multiplicity of infection of 0.001 and the control virus.Then the total protein of the infected cells was extracted and analyzed with two-dimensional difference gel electrophoresis(2 D-DIGE) 24,48,and72 hours after the infection.Potential biomarkers of H7N9 avian influenza virus infection were screened and validated via Western blot.Results Compared with that in the H1N1 pdm09 infection,the platelet-activating factor acetylhydrolaseⅠb subunit beta(PAFAH1B2) was significantly down-regulated in A549 cells 72 hours after the H7N9 avian influenza virus infection.But the expression of PAFAH1B2 showed no obvious variation in A549 cells of the blank control and infected by H1N1 pdm09 influenza virus.Conclusion PAFAH1B2 could be a biomarker for later period H7N9 avian influenza virus infection in A549 cells and the variation in PAFAH1B2 expression may be associated with clinical symptoms of H7N9 avian influenza virus infection.
Objective To analyze specific protein and screen biomarkers of H7N9 avian influenza virus infection in A549 cells by establishing a A549 cell model of H7N9 avian influenza virus and H1N1 pdm09 influenza virus(the control)infection for exploring the pathogenic mechanism of H7N9 avian influenza virus infection.Methods The A549 cells were infected by H7N9 avian influenza virus with the multiplicity of infection of 0.001 and the control virus.Then the total protein of the infected cells was extracted and analyzed with two-dimensional difference gel electrophoresis(2 D-DIGE) 24,48,and72 hours after the infection.Potential biomarkers of H7N9 avian influenza virus infection were screened and validated via Western blot.Results Compared with that in the H1N1 pdm09 infection,the platelet-activating factor acetylhydrolaseⅠb subunit beta(PAFAH1B2) was significantly down-regulated in A549 cells 72 hours after the H7N9 avian influenza virus infection.But the expression of PAFAH1B2 showed no obvious variation in A549 cells of the blank control and infected by H1N1 pdm09 influenza virus.Conclusion PAFAH1B2 could be a biomarker for later period H7N9 avian influenza virus infection in A549 cells and the variation in PAFAH1B2 expression may be associated with clinical symptoms of H7N9 avian influenza virus infection.
引文
[1]Iuliano AD,Jang Y,Jones J,et al.Increase in human infections with avian influenza A(H7N9)virus during the fifth epidemicChina,October 2016-February 2017[J].MMWR Morb Mortal Wkly Rep,2017,66(9):254-255.
[2]Jie W,Jing L,Faria NR,et al.Effect of live poultry market interventions on influenza A(H7N9)virus,Guangdong,China[J].Emerg Infect Dis,2016,22(12):2104-2112.
[3]Zeng H,Belser JA,Goldsmith CS,et al.A(H7N9)virus results in early induction of proinflammatory cytokine responses in both human lung epithelial and endothelial cells and shows increased human adaptation compared with avian H5N1 virus[J].J Virol,2015,89(8):4655-4667.
[4]Riera J,Rodriguez R,Carcedo MT,et al.Isolation and characterization of nud C from mouse macrophages,a gene implicated in the inflammatory response through the regulation of PAF-AH(I)activity[J].FEBS Lett,2007,581(16):3057-3062.
[5]Hattori M,Adachi H,Tsujimoto M,et al.Erratum:Miller-Dieker lissencephaly gene encodes a subunit of brain platelet-activating factor acetylhydrolase[J].Nature,1994,370(6486):216-218.
[6]Manya H,Aoki J,Kato H,et al.Biochemical characterization of various catalytic complexes of the brain platelet-activating factor acetylhydrolase[J].J Biol Chem,1999,274(45):31827-31832.
[7]Tjoelker LW,Stafforini DM.Platelet-activating factor acetyl-hydrolases in health and disease[J].Biochim Biophys Acta,2000,1488(1-2):102-123.
[8]To KK,Chan JF,Chen H,et al.The emergence of influenza A H7N9 in human beings 16 years after influenza A H5N1:a tale of two cities[J].Lancet Infect Dis,2013,13(9):809-821.
[9]Morrison J,Josset L,Tchitchek N,et al.H7N9 and other pathogenic avian influenza viruses elicit a three-pronged transcriptomic signature that is reminiscent of 1918 influenza virus and is associated with lethal outcome in mice[J].J Virol,2014,88(18):10556-10568.
[10]Zhou J,Wang D,Gao R,et al.Biological features of novel avian influenza A(H7N9)virus[J].Nature,2013,499(7459):500-503.
[11]Kristensson K.Avian influenza and the brain-comments on the occasion of resurrection of the Spanish flu virus[J].Brain Res Bull,2006,68(6):406-413.
[12]Maines TR,Lu XH,Erb SM,et al.Avian influenza(H5N1)viruses isolated from humans in Asia in 2004 exhibit increased virulence in mammals[J].J Virol,2005,79(18):11788-11800.
[13]Wu Y,Shen L,Ding Y,et al.Preliminary success in the characterization and management of a sudden breakout of a novel H7N9 influenza A virus[J].Int J Biol Sci,2014,10(1):109-118.
[14]王晓宏.流感病毒性脑病[J].日本医学介绍,1998,19(3):132.
[15]万献尧.流感性脑病的发病机制[J].日本医学介绍,2003,24(6):268.
[16]Bazan NG.The neuromessenger platelet-activating factor in plasticity and neurodegeneration[J].Prog Brain Res,1998,118:281-291.
[17]Shmueli O,Cahana A,Reiner O.Platelet-activating factor(PAF)acetylhydrolase activity,LIS1 expression,and seizures[J].J Neurosci Res,1999,57(2):176-184.
[18]Lindsberg PJ,Hallenbcck JM,Feuerstcin G.PAF in stroke and brain injury[J].ANN Neurol,1991,30(2):117-129.
[19]曹裕民,张雄,王丽娟.血小板活化因子及其乙酰水解酶与中枢神经系统疾病研究进展[J].中华老年心脑血管病杂志,2013,15(1):106-108.
[2]Jie W,Jing L,Faria NR,et al.Effect of live poultry market interventions on influenza A(H7N9)virus,Guangdong,China[J].Emerg Infect Dis,2016,22(12):2104-2112.
[3]Zeng H,Belser JA,Goldsmith CS,et al.A(H7N9)virus results in early induction of proinflammatory cytokine responses in both human lung epithelial and endothelial cells and shows increased human adaptation compared with avian H5N1 virus[J].J Virol,2015,89(8):4655-4667.
[4]Riera J,Rodriguez R,Carcedo MT,et al.Isolation and characterization of nud C from mouse macrophages,a gene implicated in the inflammatory response through the regulation of PAF-AH(I)activity[J].FEBS Lett,2007,581(16):3057-3062.
[5]Hattori M,Adachi H,Tsujimoto M,et al.Erratum:Miller-Dieker lissencephaly gene encodes a subunit of brain platelet-activating factor acetylhydrolase[J].Nature,1994,370(6486):216-218.
[6]Manya H,Aoki J,Kato H,et al.Biochemical characterization of various catalytic complexes of the brain platelet-activating factor acetylhydrolase[J].J Biol Chem,1999,274(45):31827-31832.
[7]Tjoelker LW,Stafforini DM.Platelet-activating factor acetyl-hydrolases in health and disease[J].Biochim Biophys Acta,2000,1488(1-2):102-123.
[8]To KK,Chan JF,Chen H,et al.The emergence of influenza A H7N9 in human beings 16 years after influenza A H5N1:a tale of two cities[J].Lancet Infect Dis,2013,13(9):809-821.
[9]Morrison J,Josset L,Tchitchek N,et al.H7N9 and other pathogenic avian influenza viruses elicit a three-pronged transcriptomic signature that is reminiscent of 1918 influenza virus and is associated with lethal outcome in mice[J].J Virol,2014,88(18):10556-10568.
[10]Zhou J,Wang D,Gao R,et al.Biological features of novel avian influenza A(H7N9)virus[J].Nature,2013,499(7459):500-503.
[11]Kristensson K.Avian influenza and the brain-comments on the occasion of resurrection of the Spanish flu virus[J].Brain Res Bull,2006,68(6):406-413.
[12]Maines TR,Lu XH,Erb SM,et al.Avian influenza(H5N1)viruses isolated from humans in Asia in 2004 exhibit increased virulence in mammals[J].J Virol,2005,79(18):11788-11800.
[13]Wu Y,Shen L,Ding Y,et al.Preliminary success in the characterization and management of a sudden breakout of a novel H7N9 influenza A virus[J].Int J Biol Sci,2014,10(1):109-118.
[14]王晓宏.流感病毒性脑病[J].日本医学介绍,1998,19(3):132.
[15]万献尧.流感性脑病的发病机制[J].日本医学介绍,2003,24(6):268.
[16]Bazan NG.The neuromessenger platelet-activating factor in plasticity and neurodegeneration[J].Prog Brain Res,1998,118:281-291.
[17]Shmueli O,Cahana A,Reiner O.Platelet-activating factor(PAF)acetylhydrolase activity,LIS1 expression,and seizures[J].J Neurosci Res,1999,57(2):176-184.
[18]Lindsberg PJ,Hallenbcck JM,Feuerstcin G.PAF in stroke and brain injury[J].ANN Neurol,1991,30(2):117-129.
[19]曹裕民,张雄,王丽娟.血小板活化因子及其乙酰水解酶与中枢神经系统疾病研究进展[J].中华老年心脑血管病杂志,2013,15(1):106-108.