【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為主要臨床表現(xiàn)的急性疾病,目前主要有H5N1、H7N7和H9N2亞型病毒可以感染人,其中H5N1感染人后病死率最高,超過(guò)50%。人H5N1病毒感染存在廣泛的免疫功能紊亂,如:機(jī)體產(chǎn)生大量的促炎癥因子和趨化因子,包括IL-1、TNF-α、IL-8、MCP-1、IP-10、MIG、RANTES等,在機(jī)體正常的免疫應(yīng)答過(guò)程中,這些因子可以保護(hù)機(jī)體,防御外來(lái)致病物,但是在特定的環(huán)境中,免疫細(xì)胞會(huì)因過(guò)度活化而攻擊正常組織,因此目前的研究推測(cè)免疫系統(tǒng)內(nèi)細(xì)胞因子的過(guò)度反應(yīng)可能與H5N1病毒的高致病性有關(guān)。呼吸道上皮細(xì)胞及單核/巨噬細(xì)胞是流感病毒感染的主要靶細(xì)胞,在病毒感染的細(xì)胞中病毒非結(jié)構(gòu)蛋白NS1含量豐富,它不僅參與了機(jī)體的免疫反應(yīng)調(diào)節(jié),還在病毒復(fù)制、細(xì)胞凋亡、抑制宿主細(xì)胞蛋白翻譯等過(guò)程中發(fā)揮重要作用。因此,本研究用含H5N1 NS1基因的重組質(zhì)粒轉(zhuǎn)染、野生病毒及重組病毒體外感染的方法,檢測(cè)人上皮細(xì)胞、單核細(xì)胞來(lái)源的巨噬細(xì)胞趨化因子的表達(dá),目的是研究我國(guó)分離的高致病性人禽流感H5N1病毒及其NS1蛋白對(duì)宿主細(xì)胞趨化因子表達(dá)的影響,進(jìn)而認(rèn)識(shí)禽流感病毒的致病機(jī)理,為其治療提供理論基礎(chǔ)。 1.人禽流感H5N1病毒能誘導(dǎo)細(xì)胞表達(dá)IP-10,但具有病毒特異性和細(xì)胞類型特異性,A/Anhui/1/2005(H5N1)誘導(dǎo)人支氣管上皮細(xì)胞BEAS-2B釋放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,這可能與禽流感H5N1亞型病毒的高致病性有關(guān)。 2.單一人流感病毒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蛋白均能下調(diào)BEAS-2B細(xì)胞內(nèi)IP-10的表達(dá)(P0.01),但三者之間下調(diào)程度無(wú)顯著差異(P0.01)。 3.以A/Puerto Rico/8/1934(H1N1)(簡(jiǎn)稱PR8-RG)為骨架含有H5N1病毒NS基因(簡(jiǎn)稱PR8-NS-RG)及插入80-84位氨基酸NS基因的重組病毒(簡(jiǎn)稱PR8-mNS-RG)誘導(dǎo)宿主細(xì)胞釋放趨化因子的表達(dá)水平有所不同,重組病毒PR8-RG感染BEAS-2B后誘導(dǎo)IP-10、IL-8、RANTES的表達(dá)水平明顯高于PR8-NS-RG、PR8-mNS-RG和未感染組(P0.05)。同樣,重組病毒PR8-RG誘導(dǎo)巨噬細(xì)胞釋放RANTES、MIG的表達(dá)水平明顯高于PR8-NS-RG、PR8-mNS-RG和未感染組(P0.05)。提示較人流感病毒H1N1,人禽流感H5N1病毒NS1基因明顯抑制趨化因子的表達(dá),且NS1基因80-84位氨基酸可能能增強(qiáng)抑制效應(yīng)。 人禽流感H5N1病毒體外感染能誘導(dǎo)趨化因子IP-10的高水平表達(dá),轉(zhuǎn)染試驗(yàn)和重組病毒感染試驗(yàn)發(fā)現(xiàn),H5N1病毒的NS1基因在拮抗細(xì)胞趨化因子反應(yīng)中表現(xiàn)了更強(qiáng)的抑制作用,提示除NS1蛋白外,可能有其它的病毒蛋白參與調(diào)節(jié)宿主對(duì)病毒的免疫應(yīng)答,也參與了H5N1病毒感染中的高炎癥。
[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- 偽, 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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R373

【參考文獻(xiàn)】

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1 龍進(jìn)學(xué);薛峰;彭宜;顧敏;劉秀梵;;H5N1亞型禽流感病毒NS第263~277位核苷酸缺失提高病毒對(duì)雞的致病力[J];微生物學(xué)報(bào);2006年02期

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本文編號(hào)：2350989