本文選題：甲型流感病毒 + 表達(dá)譜��； 參考：《北京協(xié)和醫(yī)學(xué)院》2009年博士論文

【摘要】：流感病毒是造成人類呼吸道感染的重要病原之一,流感病毒不同亞型和不同毒株不斷發(fā)生抗原漂移和抗原轉(zhuǎn)變,導(dǎo)致針對流感病毒蛋白設(shè)計(jì)的藥物由于病毒的變異迅速產(chǎn)生耐藥。從宿主角度闡明病毒與宿主之間的相互作用機(jī)制,尤其是闡明抑制或促進(jìn)病毒復(fù)制的宿主基因編碼產(chǎn)物無疑將為抗病毒研究提供新的策略和靶點(diǎn),目前已經(jīng)成為病毒學(xué)研究的熱點(diǎn)之一。鑒于此,本研究擬利用表達(dá)譜,全面分析流感病毒感染不同時(shí)間段的宿主細(xì)胞的基因組的表達(dá)變化,據(jù)此分析這些表達(dá)發(fā)生變化的基因?qū)α鞲胁《緩?fù)制的調(diào)控作用,從而為從宿主細(xì)胞基因中尋找與流感病毒復(fù)制相關(guān)的基因奠定基礎(chǔ)。 本論文首先研究了甲型流感病毒代表株A/H1N1/PR/8/34在人肺癌細(xì)胞A549上的復(fù)制特性。利用免疫熒光的方法檢測不同感染復(fù)數(shù)(MOI)在不同時(shí)間點(diǎn)的流感病毒復(fù)制情況,結(jié)果顯示隨著病毒感染量的增加,綠色熒光顯著增加,隨著時(shí)間的增多,在感染12h(h)的時(shí)候,可以明顯觀察到流感病毒的復(fù)制。MOI=1時(shí),流感病毒在A549上的復(fù)制隨時(shí)間的增多而增多,在24h時(shí),80%左右的細(xì)胞被感染,因此后續(xù)研究均選用1 MOI流感病毒感染A549細(xì)胞。然后利用1MOI的流感病毒感染A549細(xì)胞,在感染后4 h、12 h、24 h及48 h,分別提取細(xì)胞總RNA進(jìn)行表達(dá)譜分析。表達(dá)譜結(jié)果顯示,流感病毒感染4 h時(shí),只有兩個(gè)基因PRO1073、組蛋白去乙�；傅谋磉_(dá)發(fā)生了明顯的變化；感染12 h時(shí),有45個(gè)基因表達(dá)發(fā)生上調(diào),沒有發(fā)現(xiàn)下調(diào)的基因,在這45個(gè)基因中,有10個(gè)是干擾素及干擾素誘導(dǎo)表達(dá)的基因；感染24 h時(shí),表達(dá)發(fā)生變化的基因有298個(gè),其中表達(dá)上調(diào)的282個(gè)下調(diào)的16個(gè),此時(shí)干擾素上游、干擾素本身及干擾素下游調(diào)控基因均發(fā)生了明顯變化,其中流感病毒的已知的模式識(shí)別受體TLR3和RIG-I表達(dá)顯著上調(diào)；感染48h時(shí),共有265個(gè)基因表達(dá)發(fā)生變化,其中上調(diào)的有216個(gè),下調(diào)的有49個(gè)。通過熒光定量PCR對流感病毒感染細(xì)胞后表達(dá)發(fā)生上調(diào)及下調(diào)的18個(gè)基因進(jìn)行驗(yàn)證后,發(fā)現(xiàn)上調(diào)的基因與表達(dá)譜結(jié)果一致,下調(diào)的基因也與表達(dá)譜結(jié)果基本一致。然后我們對表達(dá)譜中上調(diào)及下調(diào)的基因進(jìn)行信號(hào)通路分析發(fā)現(xiàn)：表達(dá)譜中發(fā)生上調(diào)的基因大部分與干擾素相關(guān),屬于干擾素上游信號(hào)通路如MAPK通路、NF-κB通路,或者是干擾素通路本身及其誘導(dǎo)基因,而部分下調(diào)基因與糖代謝或者脂代謝相關(guān)。我們后續(xù)的研究將針對表達(dá)譜中的表達(dá)發(fā)生下調(diào)的基因進(jìn)行,擬將這些基因的表達(dá)質(zhì)粒進(jìn)行過表達(dá)后觀察對流感病毒復(fù)制的影響。 為了便于大規(guī)模篩選與流感病毒復(fù)制相關(guān)的基因,我們建立了高通量的檢測流感病毒復(fù)制的細(xì)胞內(nèi)免疫印跡法(In-Cell Western)。為了評價(jià)In-Cell Western方法,將不同MOI的流感病毒感染細(xì)胞后,與經(jīng)典的半數(shù)組織病變法(TCID50)的方法進(jìn)行比較,結(jié)果顯示In-Cell Western最低可檢測0.01 MOI的病毒感染,且與TCID50的測定結(jié)果具有較好的一致性。In-Cell Western適用于高通量檢測,具有簡便、靈敏的特點(diǎn)。 為了分析表達(dá)譜中表達(dá)發(fā)生下調(diào)基因?qū)α鞲胁《緩?fù)制是否具有調(diào)控作用,我們將表達(dá)譜中表達(dá)發(fā)生下調(diào)的基因中的34個(gè)基因進(jìn)行過表達(dá),24 h后,感染1 MOI流感病毒。24 h后通過In-Cell Western方法對病毒復(fù)制情況進(jìn)行檢查,然后挑選其中的14個(gè)有變化的基因進(jìn)行驗(yàn)證,確定8個(gè)基因?qū)α鞲胁《緩?fù)制具有抑制作用。最后選擇APOH和SULF2基因進(jìn)行劑量依賴關(guān)系分析,選擇不同的量轉(zhuǎn)染后,觀察對流感病毒復(fù)制的抑制作用。24 h后,以1 MOI的流感病毒對細(xì)胞進(jìn)行感染,用In-Cell Western進(jìn)一步驗(yàn)證這兩個(gè)基因?qū)α鞲胁《镜恼{(diào)控作用。在確定了這兩個(gè)基因?qū)α鞲胁《镜膹?fù)制與轉(zhuǎn)染的量具有劑量依賴關(guān)系后,我們接著采用同樣的方法處理細(xì)胞,利用熒光定量PCR的方法對流感病毒8個(gè)基因片段(PB2、PB1、PA、HA、NP、NA、M及NS)的vRNA、cRNA及mRNA的表達(dá)變化進(jìn)行分析。結(jié)果顯示,APOH及SULF2對流感病毒八片段的vRNA、cRNA及mRNA均有抑制作用。 綜上所述,本研究利用基因芯片技術(shù)檢測了甲型流感病毒感染A549細(xì)胞后基因轉(zhuǎn)錄的變化,發(fā)現(xiàn)了若干文獻(xiàn)里不曾報(bào)道的與流感病毒復(fù)制相關(guān)的基因,并通過對表達(dá)譜中下調(diào)的基因進(jìn)行過表達(dá),首次發(fā)現(xiàn)8個(gè)基因可能能夠抑制流感病毒復(fù)制。這些結(jié)果的取得為進(jìn)一步闡明流感病毒復(fù)制機(jī)制及闡明與流感病毒復(fù)制相關(guān)的宿主基因及其作用機(jī)制打下了基礎(chǔ)。
[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.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R373

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相關(guān)期刊論文 前1條

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