甲型流感病毒感染A549细胞的表达谱分析及相关基因功能研究

发布时间：2018-05-09 20:26

本文选题：甲型流感病毒 + 表达谱　；参考：《北京协和医学院》2009年博士论文

【摘要】：流感病毒是造成人类呼吸道感染的重要病原之一,流感病毒不同亚型和不同毒株不断发生抗原漂移和抗原转变,导致针对流感病毒蛋白设计的药物由于病毒的变异迅速产生耐药。从宿主角度阐明病毒与宿主之间的相互作用机制,尤其是阐明抑制或促进病毒复制的宿主基因编码产物无疑将为抗病毒研究提供新的策略和靶点,目前已经成为病毒学研究的热点之一。鉴于此,本研究拟利用表达谱,全面分析流感病毒感染不同时间段的宿主细胞的基因组的表达变化,据此分析这些表达发生变化的基因对流感病毒复制的调控作用,从而为从宿主细胞基因中寻找与流感病毒复制相关的基因奠定基础。本论文首先研究了甲型流感病毒代表株A/H1N1/PR/8/34在人肺癌细胞A549上的复制特性。利用免疫荧光的方法检测不同感染复数(MOI)在不同时间点的流感病毒复制情况,结果显示随着病毒感染量的增加,绿色荧光显著增加,随着时间的增多,在感染12h(h)的时候,可以明显观察到流感病毒的复制。MOI=1时,流感病毒在A549上的复制随时间的增多而增多,在24h时,80%左右的细胞被感染,因此后续研究均选用1 MOI流感病毒感染A549细胞。然后利用1MOI的流感病毒感染A549细胞,在感染后4 h、12 h、24 h及48 h,分别提取细胞总RNA进行表达谱分析。表达谱结果显示,流感病毒感染4 h时,只有两个基因PRO1073、组蛋白去乙酰化酶的表达发生了明显的变化；感染12 h时,有45个基因表达发生上调,没有发现下调的基因,在这45个基因中,有10个是干扰素及干扰素诱导表达的基因；感染24 h时,表达发生变化的基因有298个,其中表达上调的282个下调的16个,此时干扰素上游、干扰素本身及干扰素下游调控基因均发生了明显变化,其中流感病毒的已知的模式识别受体TLR3和RIG-I表达显著上调；感染48h时,共有265个基因表达发生变化,其中上调的有216个,下调的有49个。通过荧光定量PCR对流感病毒感染细胞后表达发生上调及下调的18个基因进行验证后,发现上调的基因与表达谱结果一致,下调的基因也与表达谱结果基本一致。然后我们对表达谱中上调及下调的基因进行信号通路分析发现：表达谱中发生上调的基因大部分与干扰素相关,属于干扰素上游信号通路如MAPK通路、NF-κB通路,或者是干扰素通路本身及其诱导基因,而部分下调基因与糖代谢或者脂代谢相关。我们后续的研究将针对表达谱中的表达发生下调的基因进行,拟将这些基因的表达质粒进行过表达后观察对流感病毒复制的影响。为了便于大规模筛选与流感病毒复制相关的基因,我们建立了高通量的检测流感病毒复制的细胞内免疫印迹法(In-Cell Western)。为了评价In-Cell Western方法,将不同MOI的流感病毒感染细胞后,与经典的半数组织病变法(TCID50)的方法进行比较,结果显示In-Cell Western最低可检测0.01 MOI的病毒感染,且与TCID50的测定结果具有较好的一致性。In-Cell Western适用于高通量检测,具有简便、灵敏的特点。为了分析表达谱中表达发生下调基因对流感病毒复制是否具有调控作用,我们将表达谱中表达发生下调的基因中的34个基因进行过表达,24 h后,感染1 MOI流感病毒。24 h后通过In-Cell Western方法对病毒复制情况进行检查,然后挑选其中的14个有变化的基因进行验证,确定8个基因对流感病毒复制具有抑制作用。最后选择APOH和SULF2基因进行剂量依赖关系分析,选择不同的量转染后,观察对流感病毒复制的抑制作用。24 h后,以1 MOI的流感病毒对细胞进行感染,用In-Cell Western进一步验证这两个基因对流感病毒的调控作用。在确定了这两个基因对流感病毒的复制与转染的量具有剂量依赖关系后,我们接着采用同样的方法处理细胞,利用荧光定量PCR的方法对流感病毒8个基因片段(PB2、PB1、PA、HA、NP、NA、M及NS)的vRNA、cRNA及mRNA的表达变化进行分析。结果显示,APOH及SULF2对流感病毒八片段的vRNA、cRNA及mRNA均有抑制作用。综上所述,本研究利用基因芯片技术检测了甲型流感病毒感染A549细胞后基因转录的变化,发现了若干文献里不曾报道的与流感病毒复制相关的基因,并通过对表达谱中下调的基因进行过表达,首次发现8个基因可能能够抑制流感病毒复制。这些结果的取得为进一步阐明流感病毒复制机制及阐明与流感病毒复制相关的宿主基因及其作用机制打下了基础。
[Abstract]:Influenza virus is one of the important pathogens causing human respiratory infection. The different subtypes and different strains of influenza virus continue to occur antigen drift and antigen transformation, resulting in the rapid production of drug resistance due to influenza virus protein design. The mechanism of interaction between the virus and the host from the host's angle is clarified. It is clear that the host gene encoding product that inhibits or promotes virus replication will undoubtedly provide new strategies and targets for antiviral research, and it has become one of the hotspots in virology research. In view of this, this study intends to use the expression spectrum to comprehensively analyze the changes in the expression of the genome of the host cells in different periods of influenza virus infection. The effects of these genes on the replication of influenza virus are analyzed, which lays the foundation for finding genes related to the replication of influenza virus from the host cell genes.
This thesis first studies the replication characteristics of influenza A virus representative strain A/H1N1/PR/8/34 on human lung cancer cell A549. The immunofluorescence method is used to detect the replication of influenza virus (MOI) at different time points at different time points. The results show that with the increase of virus infection, the green fluorescence increases significantly, with the increase of time. When infected with 12h (H), it is obvious that when influenza virus replication.MOI=1 is observed, the replication of influenza virus on A549 increases with the increase of time. At 24h, about 80% of the cells are infected, so the follow-up studies all choose the 1 MOI influenza virus to infect A549 cells. Then the influenza virus of 1MOI is used to infect A549 cells, and 4 h after infection, 12 h, 24 h and 48 h, respectively, to extract the total RNA expression profiles. The expression profiles showed that when influenza virus infection 4 h, only two genes PRO1073, histone deacetylase expression has changed significantly; when infection 12 h, 45 genes were up-regulated, no down regulated genes were found, in these 45 genes, 10 The gene was induced by interferon and interferon; in 24 h, 298 genes were expressed, and 16 of the 282 down-regulation were expressed. At this time interferon upstream, interferon itself and interferon downstream regulation genes were obviously changed, including the known pattern recognition receptor TLR3 and RIG-I table of the virus. When 48h was infected, there were 265 changes in gene expression, including 216 up-regulated and 49 down-regulation. 18 genes that were up and down after influenza virus infected cells were verified by fluorescence quantitative PCR, and the up regulated genes were consistent with the results of the spectrum, and the down regulated genes were also associated with the expression profiles. The results are basically the same. Then we analyze the signal pathway analysis of the up-regulated and down-regulated genes in the expression spectrum. It is found that most of the up-regulated genes in the expression spectrum are related to interferon, which belong to the upstream signal pathway such as MAPK pathway, NF- kappa B pathway, or interferon pathway and its inducible gene, while some of the genes and sugars are down regulated. Metabolism or lipid metabolism related. Our follow-up study will be aimed at genes that are down regulated in the expression spectrum, and the expression plasmid of these genes will be overexpressed to observe the impact of influenza virus replication.
In order to facilitate large-scale screening of genes associated with influenza virus replication, we have established a high throughput In-Cell Western for detecting influenza virus replication (In-Cell Western). In order to evaluate the In-Cell Western method, the virus infected cells of different MOI viruses were compared with the classical method of half tissue lesion method (TCID50). The results show that In-Cell Western can detect the lowest virus infection of 0.01 MOI, and has a good consistency with the results of TCID50..In-Cell Western is suitable for high throughput detection. It has the characteristics of simple and sensitive.
In order to analyze the regulation of the down-regulated genes expressed in the expression spectrum on influenza virus replication, we expressed 34 genes in the gene expression down-regulation in the expression spectrum. After 24 h, the infection of 1 MOI influenza virus.24 h was examined by In-Cell Western method, and then 14 of them were selected. A variant gene was tested to confirm that the 8 genes had inhibitory effect on the replication of influenza virus. Finally, APOH and SULF2 genes were selected for the dose dependence analysis. After the selection of different doses, the inhibitory effect of.24 h on the replication of influenza virus was observed, and the cells were infected with the 1 MOI virus, and In-Cell Western entered one. Step up to verify the regulation of these two genes on influenza virus. After determining the dose dependence of the two genes on the replication and transfection of influenza viruses, we then use the same method to deal with the cells and use the method of fluorescence quantitative PCR for vRNA, cRNA, and cRNA of the 8 gene fragments of influenza virus (PB2, PB1, PA, HA, NP, NA, M and NS). The expression of mRNA was analyzed. The results showed that APOH and SULF2 inhibited the vRNA, cRNA and mRNA of influenza virus eight fragment.
To sum up, the gene chip technology was used to detect the changes in gene transcription of A549 cells infected with influenza A virus, and some genes related to influenza virus replication not reported in the literature have been discovered, and the 8 genes may be found to be able to inhibit influenza virus for the first time by overexpression of the down regulated genes in the expression spectrum. These results provide a basis for further elucidation of the replication mechanism of influenza viruses and the elucidation of host genes related to the replication of influenza viruses and their mechanisms of action.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R373

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