本文選題：日本腦炎病毒　切入點(diǎn)：p53　出處：《中國(guó)農(nóng)業(yè)科學(xué)院》2011年博士論文

【摘要】：日本腦炎病毒(Japanese encephalitis virus,JEV)是一種經(jīng)蚊蟲(chóng)傳播,能引起人中樞神經(jīng)系統(tǒng)損傷的黃病毒。目前控制日本腦炎病毒的主要手段是疫苗預(yù)防和蚊蟲(chóng)清除,但是全球每年仍有近50 000的病例和近30%的死亡率。因此,仍需要開(kāi)發(fā)有效的抗病毒策略來(lái)彌補(bǔ)疫苗控制的不足。了解病毒與宿主細(xì)胞的相互作用,有利于尋找有效的抗病毒策略。p53具有抗腫瘤、DNA修復(fù)、促細(xì)胞凋亡和抗病毒等功能,許多病毒在感染過(guò)程中都與p53相互關(guān)聯(lián),但至今尚未見(jiàn)到JEV與p53相互作用的報(bào)道。為了探討p53在JEV感染中的作用,我們開(kāi)展了如下研究: 1、JEV感染降低p53的蛋白水平和轉(zhuǎn)錄活性。本研究首先用JEV(SH-JEV01株)感染A549和PIEC細(xì)胞,用間接免疫熒光(IFA)和western blot方法檢測(cè)細(xì)胞內(nèi)p53的細(xì)胞定位和蛋白水平。IFA結(jié)果顯示在感染細(xì)胞內(nèi),p53核聚集明顯減少,并且這種減少是一種普遍現(xiàn)象;western blot結(jié)果顯示,JEV感染A549細(xì)胞48h或感染PIEC細(xì)胞12h后,p53蛋白逐漸減少;將細(xì)胞質(zhì)和細(xì)胞核進(jìn)行分離,Western blot結(jié)果顯示細(xì)胞質(zhì)和胞核內(nèi)的p53均減少。通過(guò)p53熒光素酶活性分析,JEV感染后細(xì)胞內(nèi)p53活性顯著下降(p0.05);Western blot結(jié)果顯示p53靶基因IRF9的表達(dá)明顯減少。另外,IFA結(jié)果顯示,在JEV感染細(xì)胞內(nèi)NS3與p53存在細(xì)胞共定位現(xiàn)象;免疫共沉淀試驗(yàn)(co-IP)證實(shí)NS3能與p53結(jié)合。通過(guò)這部分的研究,我們首次發(fā)現(xiàn)JEV感染能降低p53蛋白水平和活性,并證實(shí)NS3蛋白與p53相互結(jié)合,提示NS3可能參與了對(duì)p53功能的影響。 2、p53抑制JEV的復(fù)制。為了觀察p53對(duì)JEV復(fù)制的影響,本研究通過(guò)RNAi技術(shù)將A549細(xì)胞內(nèi)p53進(jìn)行基因沉默,然后觀察JEV的復(fù)制。通過(guò)western blot和定量PCR分析,發(fā)現(xiàn)轉(zhuǎn)染150nM siRNA 48h后能有效的將p53基因沉默,并通過(guò)細(xì)胞活率測(cè)定和流式細(xì)胞分析,證實(shí)轉(zhuǎn)染p53 siRNA在短期內(nèi)不影響細(xì)胞的生長(zhǎng)特性及細(xì)胞周期。將JEV感染p53基因沉默的A549細(xì)胞,IFA試驗(yàn)顯示p53基因沉默組NS3陽(yáng)性的細(xì)胞數(shù)明顯增多;定量PCR試驗(yàn)表明,p53基因沉默細(xì)胞中NS3 mRNA水平顯著高于對(duì)照組(p0.01);Western blot結(jié)果表明,p53基因沉默細(xì)胞中NS3、E蛋白的表達(dá)明顯高于對(duì)照組;空斑試驗(yàn)數(shù)據(jù)顯示,p53基因沉默細(xì)胞產(chǎn)生的病毒粒子明顯多于對(duì)照組(10~100倍)。上述結(jié)果說(shuō)明,在細(xì)胞模型中p53能有效抑制JEV的復(fù)制。 3、JEV誘導(dǎo)的細(xì)胞凋亡不依賴(lài)p53。為了觀察p53在JEV誘導(dǎo)細(xì)胞凋亡中的影響,將JEV感染p53基因沉默的A549細(xì)胞,分析細(xì)胞凋亡的情況。通過(guò)臺(tái)盼藍(lán)排斥試驗(yàn),發(fā)現(xiàn)JEV感染p53基因沉默細(xì)胞后,引起更多細(xì)胞的發(fā)生死亡(p0.05);通過(guò)原位末端轉(zhuǎn)移酶標(biāo)記試驗(yàn)(TUNEL)和流式細(xì)胞術(shù)(Annexin V-PI)分析細(xì)胞凋亡情況發(fā)現(xiàn),JEV感染p53基因沉默細(xì)胞誘導(dǎo)更多細(xì)胞發(fā)生凋亡(p0.05)。從小鼠全基因表達(dá)芯片的結(jié)果中發(fā)現(xiàn)(見(jiàn)第六章),在JEV感染的p53基因敲除(p53KO)小鼠腦組織中,具有促凋亡活性的Xaf1表達(dá)水平要高于p53野生型(p53WT)小鼠89倍;定量PCR結(jié)果顯示在JEV感染的p53基因沉默細(xì)胞中,Xaf1的表達(dá)顯著高于對(duì)照組(p0.05)。上述結(jié)果說(shuō)明JEV誘導(dǎo)的細(xì)胞凋亡不依賴(lài)p53,反而由于p53的缺失,促進(jìn)了JEV誘導(dǎo)的細(xì)胞凋亡。 4、p53抑制JEV對(duì)小鼠的致病性。為了觀察p53在JEV致病性中的作用,通過(guò)皮下注射將JEV感染p53KO和p53WT小鼠。結(jié)果發(fā)現(xiàn),p53KO小鼠表現(xiàn)為癥狀出現(xiàn)早、病程短、死亡率高。通過(guò)定量PCR測(cè)定外周血中NS3 mRNA水平,結(jié)果顯示JEV在感染后第3天病毒血癥達(dá)到峰值,并且p53KO小鼠病毒血癥顯著高于野生型小鼠(p0.05)。免疫組化結(jié)果顯示,在p53KO小鼠腦組織中NS3抗原陽(yáng)性的細(xì)胞數(shù)要明顯多于p53WT小鼠。上述結(jié)果說(shuō)明在小鼠體內(nèi)p53也能抑制JEV的復(fù)制。病理切片的結(jié)果顯示,p53KO小鼠腦組織中(大腦皮層和海馬)的膠質(zhì)細(xì)胞分泌更多的炎性滲出物。ELISA結(jié)果顯示p53KO小鼠腦組織中TNF-α和IL-6的表達(dá)量要顯著高于p53WT小鼠(p0.05)。上述結(jié)果說(shuō)明,JEV感染p53KO小鼠引起更嚴(yán)重的炎癥反應(yīng)。 為了觀察JEV感染后小鼠的免疫反應(yīng),本研究通過(guò)流式細(xì)胞分析了外周血和脾臟中DC, Mφ, CD4和CD8 T細(xì)胞的比例。結(jié)果發(fā)現(xiàn),在未感染時(shí),p53KO小鼠外周血中DC細(xì)胞的比例與p53WT小鼠比較沒(méi)有顯著差別,Mφ細(xì)胞則要明顯低于p53WT小鼠;而在JEV感染后p53KO小鼠外周血中,DC和Mφ比例顯著上升,而p53WT小鼠的比例沒(méi)有顯著變化。上述結(jié)果說(shuō)明,p53影響了DC、Mφ的增殖,提示p53可能通過(guò)抑制DC和Mφ增殖,限制炎癥相關(guān)因子的產(chǎn)生;在JEV感染小鼠脾臟中,p53WT的CD4 T細(xì)胞顯著增殖,但是p53KO小鼠的CD4 T細(xì)胞的比例卻沒(méi)有變化。該結(jié)果提示p53可能通過(guò)調(diào)節(jié)CD4 T細(xì)胞的增殖,參與機(jī)體的免疫反應(yīng)。 5、小鼠腦組織全基因表達(dá)譜差異分析。為了探索p53抑制JEV復(fù)制及其致病性的機(jī)制,篩選潛在受p53調(diào)控的并參與JEV感染的基因,我們對(duì)小鼠腦組織進(jìn)行了全基因表達(dá)譜分析。通過(guò)比較p53WT和p53KO小鼠差異表達(dá)基因,我們發(fā)現(xiàn)p53KO小鼠腦組織中,一部分炎癥相關(guān)因子(趨化因子、白細(xì)胞介素和腫瘤壞死因子等)的表達(dá)上調(diào),而一些與細(xì)胞先天性免疫相關(guān)分子表達(dá)下調(diào),一些與凋亡相關(guān)的基因也有差異表達(dá)。另外還發(fā)現(xiàn):JEV感染p53基因沉默細(xì)胞后IFITM3的表達(dá)減少;在p53KO MEF中過(guò)表達(dá)p53可以誘導(dǎo)IFITM3的表達(dá);過(guò)表達(dá)p53或用5-Fu刺激能刺激IFITM3啟動(dòng)子的活性上升。這些結(jié)果說(shuō)明,IFN誘導(dǎo)的抗病毒基因IFITM3可能受到p53的調(diào)控。 綜上所述,本課題從多方面多層次研究了JEV與p53的相互作用,分析了p53對(duì)JEV復(fù)制及致病性的影響,發(fā)現(xiàn)了一些JEV感染宿主過(guò)程中潛在受p53調(diào)控的相關(guān)分子和免疫細(xì)胞,進(jìn)一步揭示了p53的先天性抗病毒免疫力和JEV的致病機(jī)理,為以后進(jìn)一步研究p53抗JEV的分子機(jī)制和開(kāi)發(fā)抗病毒藥物提供有價(jià)值的數(shù)據(jù)。
[Abstract]:Japanese encephalitis virus (Japanese encephalitis, virus, JEV) is a mosquito borne flavivirus, can cause injury of central nervous system. The main method to control Japanese encephalitis virus vaccine to prevent and remove the mosquito, but every year there are still nearly 50000 of cases and nearly 30% deaths. Therefore, still need to develop effective antiviral strategies to compensate for the lack of vaccine control. To understand the interaction between virus and host cells, to find effective antiviral strategies.P53 has anti-tumor, DNA repair, apoptosis and antiviral function, many viruses during infection and p53 are interrelated, but has yet to see the JEV interaction with p53 reports p53. In order to investigate the mechanism of JEV infection, we carried out the following research:
1, JEV infection decreased the protein levels and transcriptional activity of p53. This research first used JEV (SH-JEV01 strain) infected A549 and PIEC cells by indirect immunofluorescence (IFA) and Western blot method to detect the cellular localization of p53 in cells and the protein level of.IFA showed that in infected cells, p53 nuclear accumulation was significantly reduced, and this reduction is a common phenomenon; Western blot showed that JEV A549 cells infected with 48h or PIEC infection of 12h cells, p53 protein decreased gradually; the cytoplasm and the nucleus were isolated, Western blot results showed that the cytoplasm and nucleus of p53 were reduced by p53 luciferase activity analysis, activity of p53 cells after JEV infection significantly decreased (P0.05); Western blot showed that the expression of p53 target gene IRF9 was significantly reduced. In addition, the results of IFA showed that JEV infection in NS3 and p53 cells in cellular co localization phenomenon; CO immunoprecipitation test (co- IP) confirmed that NS3 can bind to p53. Through this part of research, we first found that JEV infection can reduce the level and activity of p53 protein, and confirm that NS3 protein is combined with p53, suggesting that NS3 may participate in the function of p53.
2, p53 inhibits replication of JEV. In order to observe the effect of p53 on JEV replication, the study by RNAi technology in A549 cells p53 gene silencing, and then observe the replication of JEV by Western blot and quantitative PCR analysis, 150nM siRNA 48h after transfection found effective p53 gene silencing, and the cell survival rate determination and flow cytometry analysis, confirmed that the transfection of p53 siRNA does not affect the growth characteristics and cell cycle of cells in a short period of time. The JEV infection of A549 cells with p53 gene silencing, IFA test showed that p53 gene silencing group NS3 positive cells increased significantly; that quantitative PCR test, NS3 mRNA level of p53 cells was significantly higher than that of gene silencing the control group (P0.01); Western blot showed that p53 gene silencing in NS3 cells, the expression of E protein was significantly higher than the control group; the empty spot test data display, virus gene silencing of p53 cells produced significantly more than the control Group (10~100 times). The results indicated that p53 could effectively inhibit the replication of JEV in the cell model.
3, JEV induced apoptosis is not dependent on p53. in order to observe the effect of p53 on JEV induced apoptosis in JEV infected A549 cells, p53 gene silencing, analysis of cell apoptosis. By trypan blue exclusion test, found that JEV infected p53 cells after gene silencing, caused by the death of more cells (P0.05) transfer; enzyme labeling test by in situ terminal (TUNEL) and flow cytometry (Annexin V-PI) cell apoptosis analysis found that induced more apoptosis JEV gene silencing cells infected with p53 (P0.05). From gene expression in mice results in chip (see Chapter sixth), the p53 gene of JEV infection at in addition to (p53KO) in the brain of mice, with the pro apoptotic activity of the expression level of Xaf1 is higher than that of the wild type p53 (p53WT) mice 89 times; quantitative PCR results showed that p53 gene silencing in JEV infected cells, the expression of Xaf1 was significantly higher than the control group (P0.05). These results suggest that JEV induced apoptosis does not depend on p53, but it promotes JEV induced apoptosis due to the absence of p53.
4, p53 inhibited the pathogenicity of JEV in mice. In order to study the effect of p53 in JEV pathogenicity, by subcutaneous injection of JEV infected p53KO mice and p53WT mice. The results showed that p53KO mice exhibited symptoms of early, short duration and high mortality. Through the quantitative determination of PCR NS3 in peripheral blood mRNA level results JEV third days after infection viremia peak viremia was significantly higher than that of p53KO mice and wild type mice (P0.05). Immunohistochemistry showed that in brain tissue of p53KO mice to NS3 antigen positive cell number was significantly higher than that in p53WT mice. These results show that JEV could also inhibit p53 replication in mice. The pathological results showed that the brain tissue of p53KO mice (cortex and hippocampus) inflammatory exudate more.ELISA results showed that the expression level of TNF- alpha and IL-6 in brain tissue of p53KO mice was significantly higher than p53WT mice secreted by glial cells (P0.05). The results suggested that JEV infection in p53KO mice caused more severe inflammatory response.
In order to observe the immune response of mice after JEV infection, this study through flow cytometric analysis of peripheral blood and spleen DC, CD4 and CD8 M. The proportion of T cells. The results showed that in the non infected p53KO mice, DC cells in peripheral blood and the proportion of p53WT mice showed no significant difference. M cells was significantly lower than that of p53WT and JEV in mice; p53KO after infection in peripheral blood of mice, significantly increased DC and M diameter ratio, while no significant changes in the proportion of p53WT mice. The results indicate that the effect of p53, DC, M. The proliferation, suggesting that p53 may inhibit the proliferation of DC and M. Limit, the levels of inflammatory cytokines in mice infected with JEV; in the spleen, CD4 T cells significantly the proliferation of p53WT, but the p53KO mouse CD4 T cells ratio did not change. The results suggest that p53 may regulate CD4 T cell proliferation, immune response in the body.
5, profiling of gene expression in brain tissue of mice. In order to explore the mechanism of inhibition of p53 JEV replication and pathogenicity, screening potential p53 regulated genes and involved in JEV infection, we conducted spectral analysis of gene expression in mouse brain tissue. The expression of genes by comparing p53WT and p53KO mice we found differences in brain tissue p53KO mice, a part of inflammatory cytokines (chemokines, interleukins and tumor necrosis factor) expression and some related cell innate immune molecule expression of some apoptosis related genes are differentially expressed. It was also found that: JEV infected p53 cells after IFITM3 gene silencing reduced expression; overexpression of p53 can induce IFITM3 in p53KO MEF; overexpression of p53 increased or stimulated by 5-Fu can stimulate the IFITM3 promoter activity. These results suggest that the anti virus IFI gene induced by IFN TM3 may be regulated by p53.
In summary, this topic from the interaction of multiple aspects and levels of JEV and p53, analyzed the effect of p53 on JEV replication and pathogenicity of JEV infection, found some potential related molecules regulated by p53 and immune cells of the host in the process, to further reveal the pathogenesis of congenital p53 anti virus immunity and JEV and to provide valuable data for the molecular mechanism and development of antiviral drugs for the further study of p53 anti JEV.

【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R373;S852.65

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