人禽流感病毒H5N1及其NS1蛋白对宿主细胞趋化因子表达影响的研究
发布时间：2018-11-23 09:00
【摘要】： 人禽流感是由甲型禽流感病毒(AIV)直接感染人而引起的以呼吸道感染为主要临床表现的急性疾病,目前主要有H5N1、H7N7和H9N2亚型病毒可以感染人,其中H5N1感染人后病死率最高,超过50%。人H5N1病毒感染存在广泛的免疫功能紊乱,如:机体产生大量的促炎症因子和趋化因子,包括IL-1、TNF-α、IL-8、MCP-1、IP-10、MIG、RANTES等,在机体正常的免疫应答过程中,这些因子可以保护机体,防御外来致病物,但是在特定的环境中,免疫细胞会因过度活化而攻击正常组织,因此目前的研究推测免疫系统内细胞因子的过度反应可能与H5N1病毒的高致病性有关。呼吸道上皮细胞及单核/巨噬细胞是流感病毒感染的主要靶细胞,在病毒感染的细胞中病毒非结构蛋白NS1含量丰富,它不仅参与了机体的免疫反应调节,还在病毒复制、细胞凋亡、抑制宿主细胞蛋白翻译等过程中发挥重要作用。因此,本研究用含H5N1 NS1基因的重组质粒转染、野生病毒及重组病毒体外感染的方法,检测人上皮细胞、单核细胞来源的巨噬细胞趋化因子的表达,目的是研究我国分离的高致病性人禽流感H5N1病毒及其NS1蛋白对宿主细胞趋化因子表达的影响,进而认识禽流感病毒的致病机理,为其治疗提供理论基础。 1.人禽流感H5N1病毒能诱导细胞表达IP-10,但具有病毒特异性和细胞类型特异性,A/Anhui/1/2005(H5N1)诱导人支气管上皮细胞BEAS-2B释放IP-10的能力高于其它H5N1病毒,且是A/Puerto Rico/8/1934(H1N1)的10倍以上,这可能与禽流感H5N1亚型病毒的高致病性有关。 2.单一人流感病毒A/Puerto Rico/8/1934(H1N1)的NS1蛋白、人禽流感病毒A/Anhui/1/2005(H5N1)NS1蛋白及插入80-84位氨基酸的人禽流感病毒NS1蛋白均能下调BEAS-2B细胞内IP-10的表达(P0.01),但三者之间下调程度无显著差异(P0.01)。 3.以A/Puerto Rico/8/1934(H1N1)(简称PR8-RG)为骨架含有H5N1病毒NS基因(简称PR8-NS-RG)及插入80-84位氨基酸NS基因的重组病毒(简称PR8-mNS-RG)诱导宿主细胞释放趋化因子的表达水平有所不同,重组病毒PR8-RG感染BEAS-2B后诱导IP-10、IL-8、RANTES的表达水平明显高于PR8-NS-RG、PR8-mNS-RG和未感染组(P0.05)。同样,重组病毒PR8-RG诱导巨噬细胞释放RANTES、MIG的表达水平明显高于PR8-NS-RG、PR8-mNS-RG和未感染组(P0.05)。提示较人流感病毒H1N1,人禽流感H5N1病毒NS1基因明显抑制趋化因子的表达,且NS1基因80-84位氨基酸可能能增强抑制效应。 人禽流感H5N1病毒体外感染能诱导趋化因子IP-10的高水平表达,转染试验和重组病毒感染试验发现,H5N1病毒的NS1基因在拮抗细胞趋化因子反应中表现了更强的抑制作用,提示除NS1蛋白外,可能有其它的病毒蛋白参与调节宿主对病毒的免疫应答,也参与了H5N1病毒感染中的高炎症。
[Abstract]:Human avian influenza is an acute disease caused by direct infection of human avian influenza A virus (AIV) with respiratory tract infection as the main clinical manifestation. At present, there are mainly H5N1 H7N7 and H9N2 subtype viruses that can infect human beings, among which H5N1 has the highest mortality rate after human infection. More than 50. Human H5N1 virus infection has a wide range of immune disorders, such as: the body produces a large number of pro-inflammatory factors and chemokines, including IL-1,TNF- 伪, IL-8,MCP-1,IP-10,MIG,RANTES, etc. In a normal immune response, these factors protect the body from foreign pathogens, but in a given environment, immune cells attack normal tissues because they are overactivated. Therefore, current studies suggest that the excessive response of cytokines in the immune system may be related to the high pathogenicity of H5N1 virus. Respiratory epithelial cells and mononuclear / macrophages are the main target cells for influenza virus infection. The viral non-structural protein NS1 is abundant in the infected cells. It not only participates in the immune response regulation of the body, but also replicates in the virus. Apoptosis, inhibition of host cell protein translation and other processes play an important role. Therefore, the expression of chemokines in macrophages derived from human epithelial cells and monocytes was detected by transfection of recombinant plasmid containing H5N1 NS1 gene and infection of wild virus and recombinant virus in vitro. The aim of this study was to study the effect of highly pathogenic human avian influenza H5N1 virus and its NS1 protein on the expression of chemokines in host cells, and to understand the pathogenesis of avian influenza virus and provide a theoretical basis for its treatment. 1. Human avian influenza H5N1 virus can induce the expression of IP-10, in cells, but it has virus specificity and cell type specificity. A/Anhui/1/2005 (H5N1) can induce BEAS-2B release of IP-10 from human bronchial epithelial cells, which is higher than that of other H5N1 viruses. And it is more than 10 times that of A/Puerto Rico/8/1934 (H1N1), which may be related to the high pathogenicity of Avian Influenza H5N1 subtype virus. 2. The NS1 protein of single human influenza virus A/Puerto Rico/8/1934 (H1N1), A/Anhui/1/2005 (H5N1) NS1 protein of human avian influenza virus (H5N1) and NS1 protein of human avian influenza virus (HAIV) inserted 80-84 amino acids could down-regulate the expression of IP-10 in BEAS-2B cells (P0.01). However, there was no significant difference in the degree of down-regulation among the three groups (P0.01). 3. Host induced by A/Puerto Rico/8/1934 (H1N1) (PR8-RG) with NS Gene of H5N1 virus (PR8-NS-RG) and NS Gene of 80-84 Amino acids inserted into NS Gene of H5N1 virus (PR8-mNS-RG) The expression level of chemokines released by cells is different. The expression level of IP-10,IL-8,RANTES induced by recombinant PR8-RG infection with BEAS-2B was significantly higher than that of PR8-NS-RG,PR8-mNS-RG and uninfected group (P0.05). Similarly, the expression of RANTES,MIG in macrophages induced by recombinant virus PR8-RG was significantly higher than that in PR8-NS-RG,PR8-mNS-RG and uninfected groups (P0.05). The results suggest that the NS1 gene of human avian influenza H5N1 virus significantly inhibits the expression of chemokines, and the 80-84 amino acid of NS1 gene may enhance the inhibitory effect. High level expression of chemokine IP-10 was induced by human avian influenza H5N1 virus infection in vitro. Transfection test and recombinant virus infection test showed that the NS1 gene of H5N1 virus had a stronger inhibitory effect on chemokine response. In addition to NS1 protein, there may be other viral proteins involved in regulating the host immune response to the virus, but also involved in the high inflammation of H5N1 virus infection.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R373
本文编号：2350989
[Abstract]:Human avian influenza is an acute disease caused by direct infection of human avian influenza A virus (AIV) with respiratory tract infection as the main clinical manifestation. At present, there are mainly H5N1 H7N7 and H9N2 subtype viruses that can infect human beings, among which H5N1 has the highest mortality rate after human infection. More than 50. Human H5N1 virus infection has a wide range of immune disorders, such as: the body produces a large number of pro-inflammatory factors and chemokines, including IL-1,TNF- 伪, IL-8,MCP-1,IP-10,MIG,RANTES, etc. In a normal immune response, these factors protect the body from foreign pathogens, but in a given environment, immune cells attack normal tissues because they are overactivated. Therefore, current studies suggest that the excessive response of cytokines in the immune system may be related to the high pathogenicity of H5N1 virus. Respiratory epithelial cells and mononuclear / macrophages are the main target cells for influenza virus infection. The viral non-structural protein NS1 is abundant in the infected cells. It not only participates in the immune response regulation of the body, but also replicates in the virus. Apoptosis, inhibition of host cell protein translation and other processes play an important role. Therefore, the expression of chemokines in macrophages derived from human epithelial cells and monocytes was detected by transfection of recombinant plasmid containing H5N1 NS1 gene and infection of wild virus and recombinant virus in vitro. The aim of this study was to study the effect of highly pathogenic human avian influenza H5N1 virus and its NS1 protein on the expression of chemokines in host cells, and to understand the pathogenesis of avian influenza virus and provide a theoretical basis for its treatment. 1. Human avian influenza H5N1 virus can induce the expression of IP-10, in cells, but it has virus specificity and cell type specificity. A/Anhui/1/2005 (H5N1) can induce BEAS-2B release of IP-10 from human bronchial epithelial cells, which is higher than that of other H5N1 viruses. And it is more than 10 times that of A/Puerto Rico/8/1934 (H1N1), which may be related to the high pathogenicity of Avian Influenza H5N1 subtype virus. 2. The NS1 protein of single human influenza virus A/Puerto Rico/8/1934 (H1N1), A/Anhui/1/2005 (H5N1) NS1 protein of human avian influenza virus (H5N1) and NS1 protein of human avian influenza virus (HAIV) inserted 80-84 amino acids could down-regulate the expression of IP-10 in BEAS-2B cells (P0.01). However, there was no significant difference in the degree of down-regulation among the three groups (P0.01). 3. Host induced by A/Puerto Rico/8/1934 (H1N1) (PR8-RG) with NS Gene of H5N1 virus (PR8-NS-RG) and NS Gene of 80-84 Amino acids inserted into NS Gene of H5N1 virus (PR8-mNS-RG) The expression level of chemokines released by cells is different. The expression level of IP-10,IL-8,RANTES induced by recombinant PR8-RG infection with BEAS-2B was significantly higher than that of PR8-NS-RG,PR8-mNS-RG and uninfected group (P0.05). Similarly, the expression of RANTES,MIG in macrophages induced by recombinant virus PR8-RG was significantly higher than that in PR8-NS-RG,PR8-mNS-RG and uninfected groups (P0.05). The results suggest that the NS1 gene of human avian influenza H5N1 virus significantly inhibits the expression of chemokines, and the 80-84 amino acid of NS1 gene may enhance the inhibitory effect. High level expression of chemokine IP-10 was induced by human avian influenza H5N1 virus infection in vitro. Transfection test and recombinant virus infection test showed that the NS1 gene of H5N1 virus had a stronger inhibitory effect on chemokine response. In addition to NS1 protein, there may be other viral proteins involved in regulating the host immune response to the virus, but also involved in the high inflammation of H5N1 virus infection.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R373
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相关期刊论文 前1条
1 龙进学;薛峰;彭宜;顾敏;刘秀梵;;H5N1亚型禽流感病毒NS第263~277位核苷酸缺失提高病毒对鸡的致病力[J];微生物学报;2006年02期
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