本文關(guān)鍵詞： 豬血凝性腦脊髓炎病毒 ATF2 表達(dá)與調(diào)控　出處：《沈陽農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：血凝性腦脊髓炎病毒主要感染1～3周齡的哺乳仔豬,常被稱為豬血凝性腦脊髓炎病毒(Porcine hemagglutinating encephalomyelitis virus,PHEV),仔豬感染后可出現(xiàn)明顯的神經(jīng)癥狀,病死率可達(dá)20%～100%。小鼠可感染PHEV,常作為感染PHEV的病理模型。本課題組前期研究表明小鼠感染PHEV后,激活轉(zhuǎn)錄因子3(Activating transcription factor 3,ATF3)的表達(dá)也增加。ATF3可作為ATF2的下游靶基因,ATF2為JNK信號(hào)通路中的一員。除JNK信號(hào)通路外,ATF3還可由P53、TGF-β/smad和c-Myc等信號(hào)通路誘導(dǎo)激活。但是前期研究中僅發(fā)現(xiàn)JNK信號(hào)通路相關(guān)因子被激活。據(jù)此推測(cè)PHEV感染小鼠后,大腦中ATF3的高表達(dá)可能是由JNK信號(hào)通路所誘導(dǎo)的。若此推論成立,作為調(diào)控ATF3的上游基因ATF2也應(yīng)呈現(xiàn)高表達(dá)。為驗(yàn)證上述假設(shè),本實(shí)驗(yàn)對(duì)感染PHEV后不同時(shí)間段的小鼠大腦中的ATF2和ATF3的表達(dá)水平進(jìn)行檢測(cè)。本實(shí)驗(yàn)首先構(gòu)建了 PHEV急性感染小鼠的模型,采用顱內(nèi)接種方式,以小鼠在接毒后第5天全部死亡的接種劑量最為最佳接種劑量。選取4周齡SPF雌性昆明小鼠30只,隨機(jī)分兩組,接毒組和對(duì)照組,每組15只。接毒組以最佳接種劑量顱內(nèi)接種PHEV,對(duì)照組以相同劑量顱內(nèi)接種PBS。于接毒后的第1天,2天,3天,4天,5天每組隨機(jī)選取3只小鼠進(jìn)行剖殺。采取大腦,一部分固定于福爾馬林溶液,另一部分凍存于液氮中。固定于福爾馬林溶液中的大腦用于病理組織學(xué)病變的觀察;凍存于液氮中的大腦,采用Real-time PCR和Western Blot法檢測(cè)ATF2和ATF3 mRNA和蛋白在大腦內(nèi)的表達(dá)情況。采用2-△△Ct相對(duì)表達(dá)量檢測(cè)ATF2、ATF3和PHEVmRNA的表達(dá)水平,采用半定量法檢測(cè)其蛋白的表達(dá)水平,并采用SPSS 17.0軟件進(jìn)行數(shù)據(jù)分析和處理。結(jié)果顯示,小鼠感染PHEV后主要表現(xiàn)為非化膿性腦炎的病理組織學(xué)變化,且隨著感染時(shí)間的延長(zhǎng),病理?yè)p傷逐漸加重。同時(shí),隨著感染時(shí)間的延長(zhǎng),ATF2、ATF3與PHEV的mRNA和蛋白表達(dá)水平均呈逐漸上升趨勢(shì)。經(jīng)相關(guān)性分析,ATF2和ATF3的表達(dá)呈正相關(guān)(p0.01),ATF2表達(dá)與PHEV之間呈正相關(guān)(p0.01),ATF3和PHEV之間表達(dá)呈正相關(guān)(p0.01)。接毒第5天達(dá)到峰值,經(jīng)One-Way ANOVA分析均具有顯著性差異(p0.01)。說明小鼠感染PHEV后大腦中的病理?yè)p傷逐步加重,且隨著ATF2的表達(dá)量的增高,ATF3表達(dá)和PHEV病毒載量也增高。小鼠感染PHEV后ATF3的高表達(dá)可能是JNK信號(hào)通路激活后由ATF2誘導(dǎo)調(diào)控的。本研究為進(jìn)一步探明PHEV感染的神經(jīng)細(xì)胞過程中ATF3高表達(dá)的機(jī)制指引了新方向,為深入解析PHEV的分子致病機(jī)制提供重要的理論依據(jù)。
[Abstract]:Hemagglutinative encephalomyelitis virus (HEV) is commonly known as porcine hemagglutinating encephalomyelitis virus PHEV, which mainly infects piglets at the age of 1 and 3 weeks. The mortality rate can reach 20: 100. Mice can be infected with pHEV, which is often used as a pathological model of PHEV infection. Our previous study showed that mice infected with PHEV, The expression of activator transcription factor 3 (ATF3) was also increased. ATF3 could be used as a downstream target gene of ATF2, ATF2 was a member of JNK signaling pathway. Besides JNK signaling pathway, ATF3 could also be induced by P53 TGF- 尾 -smad and c-Myc signaling pathway, but in previous studies, it could also be activated by signal pathways such as P53 TGF- 尾 -smad and c-Myc. Only JNK signaling pathway related factors were found to be activated. It is speculated that after PHEV infection in mice, The high expression of ATF3 in the brain may be induced by the JNK signaling pathway. If this corollary is true, the upstream gene ATF2, which regulates ATF3, should also be overexpressed. The expression levels of ATF2 and ATF3 in the brain of mice infected with PHEV at different time periods were detected. Firstly, a model of mice with acute PHEV infection was established, and intracranial inoculation was used to detect the expression of ATF2 and ATF3 in the brain of mice. 30 SPF female Kunming mice aged 4 weeks were randomly divided into two groups. 15 mice in each group were inoculated with pHEV at the best inoculation dose, and PBSs were inoculated in the same dose in the control group. 3 mice in each group were randomly selected for dissection on the first day, 2 days and 4 days and 5 days after inoculation. One part is fixed in a formalin solution, the other part is frozen in liquid nitrogen. The brain fixed in a formalin solution is used to observe pathological changes; the brain is frozen in liquid nitrogen. Real-time PCR and Western Blot were used to detect the expression of ATF2 and ATF3 mRNA and protein in the brain, the relative expression of ATF2Ct and the expression of ATF3 and PHEVmRNA were detected by the relative expression of 2Ct, and the expression levels of ATF2pATF3 and PHEVmRNA were detected by semi-quantitative method. SPSS 17.0 software was used to analyze and process the data. The results showed that the histopathological changes of non-suppurative encephalitis occurred mainly in mice infected with PHEV, and the pathological damage was aggravated with the prolongation of infection time. The expression of ATF2 and ATF3 were positively correlated with the expression of PHEV, and the expression of ATF3 and PHEV were positively correlated with the expression of PHEV and the expression of ATF3. The expression of ATF3 and PHEV increased gradually with the prolongation of the infection time, and the expression of ATF2 and ATF3 were positively correlated with the expression of AATF3 and PHEV, the correlation analysis showed that the expression of ATF2 and ATF3 were positively correlated with the expression of PHEV and the expression of ATF3 was positively correlated with the expression of PHEV. The poison reached its peak on the 5th day, The results of One-Way ANOVA analysis showed that the pathological damage in the brain of mice infected with PHEV increased gradually. With the increase of ATF2 expression, ATF3 expression and PHEV viral load also increased. The high expression of ATF3 in mice infected with PHEV may be regulated by ATF2 induced by JNK signal pathway. This study is to further explore the fine nerve of PHEV infection. The mechanism of high expression of ATF3 in cellular process indicates a new direction. It provides an important theoretical basis for further analysis of molecular pathogenesis of PHEV.
【學(xué)位授予單位】：沈陽農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S858.28

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本文編號(hào)：1504314