小鼠感染猪血凝性脑脊髓炎病毒后大脑内ATF2和ATF3表达的动态变化

发布时间：2018-02-12 00:00

本文关键词： 猪血凝性脑脊髓炎病毒 ATF2 表达与调控　出处：《沈阳农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：血凝性脑脊髓炎病毒主要感染1～3周龄的哺乳仔猪,常被称为猪血凝性脑脊髓炎病毒(Porcine hemagglutinating encephalomyelitis virus,PHEV),仔猪感染后可出现明显的神经症状,病死率可达20%～100%。小鼠可感染PHEV,常作为感染PHEV的病理模型。本课题组前期研究表明小鼠感染PHEV后,激活转录因子3(Activating transcription factor 3,ATF3)的表达也增加。ATF3可作为ATF2的下游靶基因,ATF2为JNK信号通路中的一员。除JNK信号通路外,ATF3还可由P53、TGF-β/smad和c-Myc等信号通路诱导激活。但是前期研究中仅发现JNK信号通路相关因子被激活。据此推测PHEV感染小鼠后,大脑中ATF3的高表达可能是由JNK信号通路所诱导的。若此推论成立,作为调控ATF3的上游基因ATF2也应呈现高表达。为验证上述假设,本实验对感染PHEV后不同时间段的小鼠大脑中的ATF2和ATF3的表达水平进行检测。本实验首先构建了 PHEV急性感染小鼠的模型,采用颅内接种方式,以小鼠在接毒后第5天全部死亡的接种剂量最为最佳接种剂量。选取4周龄SPF雌性昆明小鼠30只,随机分两组,接毒组和对照组,每组15只。接毒组以最佳接种剂量颅内接种PHEV,对照组以相同剂量颅内接种PBS。于接毒后的第1天,2天,3天,4天,5天每组随机选取3只小鼠进行剖杀。采取大脑,一部分固定于福尔马林溶液,另一部分冻存于液氮中。固定于福尔马林溶液中的大脑用于病理组织学病变的观察;冻存于液氮中的大脑,采用Real-time PCR和Western Blot法检测ATF2和ATF3 mRNA和蛋白在大脑内的表达情况。采用2-△△Ct相对表达量检测ATF2、ATF3和PHEVmRNA的表达水平,采用半定量法检测其蛋白的表达水平,并采用SPSS 17.0软件进行数据分析和处理。结果显示,小鼠感染PHEV后主要表现为非化脓性脑炎的病理组织学变化,且随着感染时间的延长,病理损伤逐渐加重。同时,随着感染时间的延长,ATF2、ATF3与PHEV的mRNA和蛋白表达水平均呈逐渐上升趋势。经相关性分析,ATF2和ATF3的表达呈正相关(p0.01),ATF2表达与PHEV之间呈正相关(p0.01),ATF3和PHEV之间表达呈正相关(p0.01)。接毒第5天达到峰值,经One-Way ANOVA分析均具有显著性差异(p0.01)。说明小鼠感染PHEV后大脑中的病理损伤逐步加重,且随着ATF2的表达量的增高,ATF3表达和PHEV病毒载量也增高。小鼠感染PHEV后ATF3的高表达可能是JNK信号通路激活后由ATF2诱导调控的。本研究为进一步探明PHEV感染的神经细胞过程中ATF3高表达的机制指引了新方向,为深入解析PHEV的分子致病机制提供重要的理论依据。
[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.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.28

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