p53在日本脑炎病毒复制及其致病性中的作用

发布时间：2018-03-28 17:50

  本文选题：日本脑炎病毒　切入点：p53　出处：《中国农业科学院》2011年博士论文

【摘要】：日本脑炎病毒(Japanese encephalitis virus,JEV)是一种经蚊虫传播,能引起人中枢神经系统损伤的黄病毒。目前控制日本脑炎病毒的主要手段是疫苗预防和蚊虫清除,但是全球每年仍有近50 000的病例和近30%的死亡率。因此,仍需要开发有效的抗病毒策略来弥补疫苗控制的不足。了解病毒与宿主细胞的相互作用,有利于寻找有效的抗病毒策略。p53具有抗肿瘤、DNA修复、促细胞凋亡和抗病毒等功能,许多病毒在感染过程中都与p53相互关联,但至今尚未见到JEV与p53相互作用的报道。为了探讨p53在JEV感染中的作用,我们开展了如下研究: 1、JEV感染降低p53的蛋白水平和转录活性。本研究首先用JEV(SH-JEV01株)感染A549和PIEC细胞,用间接免疫荧光(IFA)和western blot方法检测细胞内p53的细胞定位和蛋白水平。IFA结果显示在感染细胞内,p53核聚集明显减少,并且这种减少是一种普遍现象;western blot结果显示,JEV感染A549细胞48h或感染PIEC细胞12h后,p53蛋白逐渐减少;将细胞质和细胞核进行分离,Western blot结果显示细胞质和胞核内的p53均减少。通过p53荧光素酶活性分析,JEV感染后细胞内p53活性显著下降(p0.05);Western blot结果显示p53靶基因IRF9的表达明显减少。另外,IFA结果显示,在JEV感染细胞内NS3与p53存在细胞共定位现象;免疫共沉淀试验(co-IP)证实NS3能与p53结合。通过这部分的研究,我们首次发现JEV感染能降低p53蛋白水平和活性,并证实NS3蛋白与p53相互结合,提示NS3可能参与了对p53功能的影响。 2、p53抑制JEV的复制。为了观察p53对JEV复制的影响,本研究通过RNAi技术将A549细胞内p53进行基因沉默,然后观察JEV的复制。通过western blot和定量PCR分析,发现转染150nM siRNA 48h后能有效的将p53基因沉默,并通过细胞活率测定和流式细胞分析,证实转染p53 siRNA在短期内不影响细胞的生长特性及细胞周期。将JEV感染p53基因沉默的A549细胞,IFA试验显示p53基因沉默组NS3阳性的细胞数明显增多;定量PCR试验表明,p53基因沉默细胞中NS3 mRNA水平显著高于对照组(p0.01);Western blot结果表明,p53基因沉默细胞中NS3、E蛋白的表达明显高于对照组;空斑试验数据显示,p53基因沉默细胞产生的病毒粒子明显多于对照组(10~100倍)。上述结果说明,在细胞模型中p53能有效抑制JEV的复制。 3、JEV诱导的细胞凋亡不依赖p53。为了观察p53在JEV诱导细胞凋亡中的影响,将JEV感染p53基因沉默的A549细胞,分析细胞凋亡的情况。通过台盼蓝排斥试验,发现JEV感染p53基因沉默细胞后,引起更多细胞的发生死亡(p0.05);通过原位末端转移酶标记试验(TUNEL)和流式细胞术(Annexin V-PI)分析细胞凋亡情况发现,JEV感染p53基因沉默细胞诱导更多细胞发生凋亡(p0.05)。从小鼠全基因表达芯片的结果中发现(见第六章),在JEV感染的p53基因敲除(p53KO)小鼠脑组织中,具有促凋亡活性的Xaf1表达水平要高于p53野生型(p53WT)小鼠89倍;定量PCR结果显示在JEV感染的p53基因沉默细胞中,Xaf1的表达显著高于对照组(p0.05)。上述结果说明JEV诱导的细胞凋亡不依赖p53,反而由于p53的缺失,促进了JEV诱导的细胞凋亡。 4、p53抑制JEV对小鼠的致病性。为了观察p53在JEV致病性中的作用,通过皮下注射将JEV感染p53KO和p53WT小鼠。结果发现,p53KO小鼠表现为症状出现早、病程短、死亡率高。通过定量PCR测定外周血中NS3 mRNA水平,结果显示JEV在感染后第3天病毒血症达到峰值,并且p53KO小鼠病毒血症显著高于野生型小鼠(p0.05)。免疫组化结果显示,在p53KO小鼠脑组织中NS3抗原阳性的细胞数要明显多于p53WT小鼠。上述结果说明在小鼠体内p53也能抑制JEV的复制。病理切片的结果显示,p53KO小鼠脑组织中(大脑皮层和海马)的胶质细胞分泌更多的炎性渗出物。ELISA结果显示p53KO小鼠脑组织中TNF-α和IL-6的表达量要显著高于p53WT小鼠(p0.05)。上述结果说明,JEV感染p53KO小鼠引起更严重的炎症反应。 为了观察JEV感染后小鼠的免疫反应,本研究通过流式细胞分析了外周血和脾脏中DC, Mφ, CD4和CD8 T细胞的比例。结果发现,在未感染时,p53KO小鼠外周血中DC细胞的比例与p53WT小鼠比较没有显著差别,Mφ细胞则要明显低于p53WT小鼠;而在JEV感染后p53KO小鼠外周血中,DC和Mφ比例显著上升,而p53WT小鼠的比例没有显著变化。上述结果说明,p53影响了DC、Mφ的增殖,提示p53可能通过抑制DC和Mφ增殖,限制炎症相关因子的产生;在JEV感染小鼠脾脏中,p53WT的CD4 T细胞显著增殖,但是p53KO小鼠的CD4 T细胞的比例却没有变化。该结果提示p53可能通过调节CD4 T细胞的增殖,参与机体的免疫反应。 5、小鼠脑组织全基因表达谱差异分析。为了探索p53抑制JEV复制及其致病性的机制,筛选潜在受p53调控的并参与JEV感染的基因,我们对小鼠脑组织进行了全基因表达谱分析。通过比较p53WT和p53KO小鼠差异表达基因,我们发现p53KO小鼠脑组织中,一部分炎症相关因子(趋化因子、白细胞介素和肿瘤坏死因子等)的表达上调,而一些与细胞先天性免疫相关分子表达下调,一些与凋亡相关的基因也有差异表达。另外还发现:JEV感染p53基因沉默细胞后IFITM3的表达减少;在p53KO MEF中过表达p53可以诱导IFITM3的表达;过表达p53或用5-Fu刺激能刺激IFITM3启动子的活性上升。这些结果说明,IFN诱导的抗病毒基因IFITM3可能受到p53的调控。 综上所述,本课题从多方面多层次研究了JEV与p53的相互作用,分析了p53对JEV复制及致病性的影响,发现了一些JEV感染宿主过程中潜在受p53调控的相关分子和免疫细胞,进一步揭示了p53的先天性抗病毒免疫力和JEV的致病机理,为以后进一步研究p53抗JEV的分子机制和开发抗病毒药物提供有价值的数据。
[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.

【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R373;S852.65

【参考文献】

相关期刊论文 前1条

1 邓绪芳;史子学;邱亚峰;沈阳;邵东华;马志永;;流行性乙型脑炎病原生态学的研究概况[J];动物医学进展;2011年02期

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