ATF3在猪血凝性脑脊髓炎病毒感染神经细胞过程中对细胞周期和凋亡的影响

发布时间：2018-03-19 04:05

  本文选题：猪血凝性脑脊髓炎病毒　切入点：ATF3　出处：《沈阳农业大学》2017年硕士论文　论文类型：学位论文

【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)引起的,主要感染3周龄的仔猪,临床以呕吐衰竭和明显的神经症状为特征。前期研究结果表明,小鼠感染PHEV后大脑中ATF3呈高表达,为了探明ATF3在神经细胞感染PHEV后ATF3是否呈现高表达,以及其高表达的机制,本实验对神经细胞感染PHEV前后ATF3的表达进行了检测,同时探索JNK信号通路在猪血凝性脑脊髓炎病毒感染神经细胞过程中对ATF3表达的影响。本试验以N2a细胞作为研究PHEV感染神经细胞的模型,根据试验要求,设置4组:对照组、接毒组、JNK抑制剂组和JNK抑制剂接毒组,每组3个重复。于六孔板中培养N2a细胞到单层,以吸附法接种PHEV,然后分别在2h、12h、24h时提取细胞总RNA和总蛋白,通过荧光定量PCR和蛋白质印迹法(Western Blot)分别测定PHEV感染前后及JNK抑制剂作用前后N2a细胞中ATF3 mRNA和蛋白的表达量。同时设置6组:对照组、接毒组、DMSO组、DMSO接毒组、JNK抑制剂组和JNK抑制剂接毒组,每组3个重复,通过Annexin V-FITC/PI双染法和PI染色法检测12h和24h的细胞凋亡和细胞周期,以测定JNK抑制剂作用前后对PHEV感染的神经细胞的凋亡和细胞周期的影响。结果表明,ATF3基因在正常N2a细胞中的处于低表达,随着PHEV mRNA和蛋白表达量的降低或升高ATF3 mRNA和蛋白的表达量也随着降低或升高,两者之间的变化趋势一致且呈正相关;JNK抑制剂作用N2a细胞后,ATF3的mRNA和蛋白表达量明显降低。通过Annexin V-FITC/PI双染法标记凋亡细胞,再经流式细胞仪进行检测,结果发现与对照组相比,JNK抑制剂作用后接毒组细胞凋亡率明显上升,差异极显著(P0.01),此时ATF3表达量降低,说明ATF3在细胞内起到抑制凋亡的作用;同时,与对照组相比,JNK抑制剂作用后接毒组细胞周期发生阻滞。N2a细胞感染PHEV后,ATF3的表达量增高。JNK抑制剂作用于N2a细胞后,ATF3表达量下降,细胞凋亡率上升,细胞周期阻滞,DNA合成受阻,细胞活力下降,抗病毒能力下降。表明神经细胞感染PHEV后JNK信号通路可调控ATF3的表达,进而影响神经细胞凋亡和细胞周期。
[Abstract]:Porcine hemagglutinating encephalomyelitis virus (Porcine hemagglutinating encephalomyelitis virus PHEV) is the main cause of porcine hemagglutinative encephalomyelitis. It mainly infects 3-week-old piglets and is characterized by vomiting failure and obvious neurological symptoms. The expression of ATF3 was high in the brain of mice infected with PHEV. In order to find out whether the expression of ATF3 was high after PHEV infection and the mechanism of its high expression, the expression of ATF3 before and after the infection of PHEV in nerve cells was detected in this experiment. At the same time, the effect of JNK signal pathway on the expression of ATF3 in the course of infection of porcine hemagglutinating encephalomyelitis virus was explored. N2a cells were used as the model of PHEV infection nerve cells. According to the requirements of the experiment, four groups were set up: control group. N2a cells were cultured in a six-well plate to a monolayer, then inoculated with pHEV by adsorption method. The total RNA and total protein were extracted at 2 h and 12 h after inoculation, respectively. The expression of ATF3 mRNA and protein in N2a cells before and after PHEV infection and before and after treatment with JNK inhibitor were measured by fluorescence quantitative PCR and Western blot respectively. The apoptosis and cell cycle were detected by Annexin V-FITC / Pi double staining and Pi staining at 12h and 24h by Annexin V-FITC / Pi double staining. To determine the effect of JNK inhibitor on apoptosis and cell cycle of PHEV infected neurons, the results showed that the expression of ATF3 gene was low in normal N2a cells. With the decrease or increase of PHEV mRNA and protein expression, the expression of ATF3 mRNA and protein decreased or increased. The expression of mRNA and protein of ATF3 in N2a cells was significantly decreased after treated with JNK inhibitor. The apoptotic cells were labeled with Annexin V-FITC / Pi double staining and detected by flow cytometry. The results showed that compared with the control group, the apoptosis rate of the treated group was significantly higher than that of the control group, and the difference was very significant (P 0.01). At the same time, the expression of ATF3 was decreased, which indicated that ATF3 played a role in inhibiting apoptosis in the cells, at the same time, Compared with the control group, the expression of ATF3 in N2a cells infected with PHEV was increased. The expression of ATF3 in N2a cells decreased, the apoptosis rate increased, and the cell cycle blocked the synthesis of ATF3 in N2a cells. The decrease of cell viability and the decrease of anti-virus ability suggest that JNK signaling pathway can regulate the expression of ATF3 and then affect the apoptosis and cell cycle of neurons after infection with 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】：S852.65
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本文编号：1632727