本文選題：豬血凝性腦脊髓炎病毒 + ATF3�。� 參考：《沈陽農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：豬血凝性腦脊髓炎是由豬血凝性腦脊髓炎病毒(Porcine hemagglutinating encephalomyelitis virus, PHEV)引起仔豬的一種急性、高度接觸性傳染病,主要侵害1～3周齡哺乳期的仔豬,臨床以仔豬嘔吐、衰竭和/或明顯的神經(jīng)癥狀為主要特征,死亡率達(dá)20%～100%,給養(yǎng)殖業(yè)帶來巨大的經(jīng)濟(jì)損失。PHEV具有嗜神經(jīng)性,主要由感染部位經(jīng)外周神經(jīng)向中樞神經(jīng)系統(tǒng)傳播,引起大腦呈現(xiàn)非化膿性腦脊髓炎的病理性損傷。MX1蛋白是一種抗病毒蛋白,由Ⅰ型干擾素(IFNα/β)誘導(dǎo)宿主細(xì)胞產(chǎn)生的,具有廣譜的抗病毒作用。ATF3是激活轉(zhuǎn)錄因子家族成員,是轉(zhuǎn)錄調(diào)控、細(xì)胞凋亡、細(xì)胞分裂和生存的一個重要調(diào)控因子,可以使細(xì)胞增殖加速,與病毒的復(fù)制密切相關(guān)。本實(shí)驗(yàn)室前期研究表明,小鼠大腦皮層及神經(jīng)瘤細(xì)胞N2a感染PHEV后,ATF3基因與MX1基因均呈差異表達(dá)。但這兩種基因差異表達(dá)是否與病毒感染有直接的關(guān)系,其表達(dá)水平與PHEV病毒載量之間是否有相關(guān)性尚不明確。因此,本研究旨在通過檢測PHEV感染后大腦中ATF3和MX1的表達(dá)水平,探索ATF3、MX1表達(dá)水平和PHEV病毒載量之間的關(guān)系,為闡明抗病毒蛋白和活性轉(zhuǎn)錄因子的作用機(jī)理奠定一定的理論基礎(chǔ)。方法：建立PHEV急性感染小鼠模型,用0.1mL的PHEV(103TCID50/0.1mL)以顱內(nèi)接種方式感染5周齡的雌性健康昆明小鼠,使小鼠在第5d全部死亡。之后將30只小鼠隨機(jī)分為病毒組和對照組,每組各15只,病毒組顱內(nèi)注射0.1mL的PHEV,對照組接種相同劑量的PBS。在感染后第1～5d每天每組分別隨機(jī)選3只小鼠剖殺,取大腦一部分于液氮中保存,進(jìn)行熒光定量PCR；另一部分大腦固定于福爾馬林溶液中,進(jìn)行H.E.染色和免疫組織化學(xué)試驗(yàn)。結(jié)果發(fā)現(xiàn),接毒后第1～2d的小鼠精神狀態(tài)及飲食無明顯異常；接毒后第3d小鼠出現(xiàn)明顯的神經(jīng)癥狀,如弓背,兩前肢呈“彈鋼琴樣”上下擺動；接毒后第5d小鼠癱瘓,抽搐死亡；對照組小鼠無異常反應(yīng)。剖檢發(fā)現(xiàn)接毒組小鼠大腦充血水腫；組織病理學(xué)觀察發(fā)現(xiàn)接毒組小鼠大腦呈典型的非化膿性腦炎變化,如神經(jīng)元固縮、壞死,小膠質(zhì)細(xì)胞增多,衛(wèi)星現(xiàn)象及“血管套”形成。Real-time PCR結(jié)果顯示,接毒組小鼠大腦ATF3和MX1基因表達(dá)水平與PHEV病毒載量均升高,且ATF3和MX1基因表達(dá)水平與PHEV的病毒載量呈現(xiàn)正相關(guān)關(guān)系,相關(guān)性極顯著(p0.01)。對不同感染時間段小鼠大腦PHEV病毒載量和ATF3基因表達(dá)水平進(jìn)行比較,結(jié)果顯示,對照組、感染第1～4d小鼠與第5d小鼠之間病毒載量和ATF3基因表達(dá)水平均呈現(xiàn)顯著差異(p0.05)。免疫組化結(jié)果顯示,小鼠感染病毒后大腦ATF3、MX1蛋白及PHEV的胞漿中均有棕黃色陽性信號,且大腦中ATF3蛋白、MX1蛋白表達(dá)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- 偽 / 尾 of interferon type I (IFN- 偽 / 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.
【學(xué)位授予單位】：沈陽農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S858.28

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