小鼠感染猪血凝性脑脊髓炎病毒后对大脑ATF3与MX1表达的影响

发布时间：2018-04-19 11:07

本文选题：猪血凝性脑脊髓炎病毒 + ATF3　；参考：《沈阳农业大学》2016年硕士论文

【摘要】：猪血凝性脑脊髓炎是由猪血凝性脑脊髓炎病毒(Porcine hemagglutinating encephalomyelitis virus, PHEV)引起仔猪的一种急性、高度接触性传染病,主要侵害1～3周龄哺乳期的仔猪,临床以仔猪呕吐、衰竭和/或明显的神经症状为主要特征,死亡率达20%～100%,给养殖业带来巨大的经济损失。PHEV具有嗜神经性,主要由感染部位经外周神经向中枢神经系统传播,引起大脑呈现非化脓性脑脊髓炎的病理性损伤。MX1蛋白是一种抗病毒蛋白,由Ⅰ型干扰素(IFNα/β)诱导宿主细胞产生的,具有广谱的抗病毒作用。ATF3是激活转录因子家族成员,是转录调控、细胞凋亡、细胞分裂和生存的一个重要调控因子,可以使细胞增殖加速,与病毒的复制密切相关。本实验室前期研究表明,小鼠大脑皮层及神经瘤细胞N2a感染PHEV后,ATF3基因与MX1基因均呈差异表达。但这两种基因差异表达是否与病毒感染有直接的关系,其表达水平与PHEV病毒载量之间是否有相关性尚不明确。因此,本研究旨在通过检测PHEV感染后大脑中ATF3和MX1的表达水平,探索ATF3、MX1表达水平和PHEV病毒载量之间的关系,为阐明抗病毒蛋白和活性转录因子的作用机理奠定一定的理论基础。方法：建立PHEV急性感染小鼠模型,用0.1mL的PHEV(103TCID50/0.1mL)以颅内接种方式感染5周龄的雌性健康昆明小鼠,使小鼠在第5d全部死亡。之后将30只小鼠随机分为病毒组和对照组,每组各15只,病毒组颅内注射0.1mL的PHEV,对照组接种相同剂量的PBS。在感染后第1～5d每天每组分别随机选3只小鼠剖杀,取大脑一部分于液氮中保存,进行荧光定量PCR；另一部分大脑固定于福尔马林溶液中,进行H.E.染色和免疫组织化学试验。结果发现,接毒后第1～2d的小鼠精神状态及饮食无明显异常；接毒后第3d小鼠出现明显的神经症状,如弓背,两前肢呈“弹钢琴样”上下摆动；接毒后第5d小鼠瘫痪,抽搐死亡；对照组小鼠无异常反应。剖检发现接毒组小鼠大脑充血水肿；组织病理学观察发现接毒组小鼠大脑呈典型的非化脓性脑炎变化,如神经元固缩、坏死,小胶质细胞增多,卫星现象及“血管套”形成。Real-time PCR结果显示,接毒组小鼠大脑ATF3和MX1基因表达水平与PHEV病毒载量均升高,且ATF3和MX1基因表达水平与PHEV的病毒载量呈现正相关关系,相关性极显著(p0.01)。对不同感染时间段小鼠大脑PHEV病毒载量和ATF3基因表达水平进行比较,结果显示,对照组、感染第1～4d小鼠与第5d小鼠之间病毒载量和ATF3基因表达水平均呈现显著差异(p0.05)。免疫组化结果显示,小鼠感染病毒后大脑ATF3、MX1蛋白及PHEV的胞浆中均有棕黄色阳性信号,且大脑中ATF3蛋白、MX1蛋白表达量和PHEV均增加。
[Abstract]:Porcine hemagglutinating encephalomyelitis virus (pHEV) is an acute and highly contact infectious disease in piglets, which mainly affects piglets during lactation at the age of 1 to 3 weeks.Failure and / or obvious neurological symptoms were the main characteristics, and the mortality rate was 20% 100%. PHEV caused enormous economic losses to the aquaculture industry. PHEV was neurophilic, mainly transmitted from the infected site through the peripheral nerve to the central nervous system (CNS).Pathological injury. MX1 protein is an antiviral protein that is produced by IFN- 伪 / 尾 of interferon type I (IFN- 伪 / 尾). ATF3 is a member of activator transcription factor family.It is an important regulatory factor for transcription regulation, apoptosis, cell division and survival. It can accelerate cell proliferation and is closely related to virus replication.Our previous study showed that the expression of ATF3 gene was different from that of MX1 gene after N2a infection in mouse cerebral cortex and neuroma cells.However, whether the differential expression of these two genes is directly related to viral infection, and whether there is a correlation between the expression level and the viral load of PHEV is not clear.Therefore, the purpose of this study was to investigate the relationship between ATF3mX1 expression level and PHEV viral load by detecting the expression of ATF3 and MX1 in the brain after PHEV infection, and to lay a theoretical foundation for elucidating the mechanism of antiviral protein and active transcription factor.Methods: the mice model of acute PHEV infection was established. The female healthy Kunming mice of 5 weeks old were inoculated with 0.1mL 's pHEV 103TCID 50 / 0.1mL, and all the mice died on the 5th day.After that, 30 mice were randomly divided into virus group and control group with 15 mice in each group. The virus group was injected with pHEV of 0.1mL, and the control group was inoculated with the same dose of PBSs.On the first day after infection, 3 mice in each group were randomly selected to be killed. One part of brain was preserved in liquid nitrogen for fluorescence quantitative PCR, the other part of brain was fixed in formalin solution for H. E.Staining and immunohistochemistry.The results showed that there was no obvious abnormality in the mental state and diet of the mice on the 1st and 2nd day after exposure, and the mice developed obvious neurological symptoms on the 3rd day, such as the back of the bow, the forelimbs swinging up and down like playing the piano, and the mice were paralyzed and died of convulsions on the 5th day.There was no abnormal reaction in the control group.Histopathological observation showed that the mice in the exposed group showed typical changes of non-suppurative encephalitis, such as pyknosis, necrosis and increase of microglia.The results of satellite phenomenon and vasculoid formation. Real-time PCR showed that the expression level of ATF3 and MX1 genes and the viral load of PHEV increased in the exposed group, and there was a positive correlation between the expression level of ATF3 and MX1 genes and the viral load of PHEV, and the correlation was very significant (p0.01).The amount of PHEV virus and the expression level of ATF3 gene in the brain of mice at different infection periods were compared. The results showed that there were significant differences in viral load and ATF3 gene expression level between the control group and the mice on the 1st day and 5th day of infection.The results of immunohistochemistry showed that there were brown positive signals in the cytoplasm of ATF3mX1 protein and PHEV, and the expression of ATF3 protein MX1 protein and PHEV increased in the brain of mice infected with the virus.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S858.28

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