本文選題：豬血凝性腦脊髓炎病毒　切入點：ATF3　出處：《沈陽農業(yè)大學》2017年碩士論文　論文類型：學位論文

【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)引起的,主要感染3周齡的仔豬,臨床以嘔吐衰竭和明顯的神經癥狀為特征。前期研究結果表明,小鼠感染PHEV后大腦中ATF3呈高表達,為了探明ATF3在神經細胞感染PHEV后ATF3是否呈現高表達,以及其高表達的機制,本實驗對神經細胞感染PHEV前后ATF3的表達進行了檢測,同時探索JNK信號通路在豬血凝性腦脊髓炎病毒感染神經細胞過程中對ATF3表達的影響。本試驗以N2a細胞作為研究PHEV感染神經細胞的模型,根據試驗要求,設置4組:對照組、接毒組、JNK抑制劑組和JNK抑制劑接毒組,每組3個重復。于六孔板中培養(yǎng)N2a細胞到單層,以吸附法接種PHEV,然后分別在2h、12h、24h時提取細胞總RNA和總蛋白,通過熒光定量PCR和蛋白質印跡法(Western Blot)分別測定PHEV感染前后及JNK抑制劑作用前后N2a細胞中ATF3 mRNA和蛋白的表達量。同時設置6組:對照組、接毒組、DMSO組、DMSO接毒組、JNK抑制劑組和JNK抑制劑接毒組,每組3個重復,通過Annexin V-FITC/PI雙染法和PI染色法檢測12h和24h的細胞凋亡和細胞周期,以測定JNK抑制劑作用前后對PHEV感染的神經細胞的凋亡和細胞周期的影響。結果表明,ATF3基因在正常N2a細胞中的處于低表達,隨著PHEV mRNA和蛋白表達量的降低或升高ATF3 mRNA和蛋白的表達量也隨著降低或升高,兩者之間的變化趨勢一致且呈正相關;JNK抑制劑作用N2a細胞后,ATF3的mRNA和蛋白表達量明顯降低。通過Annexin V-FITC/PI雙染法標記凋亡細胞,再經流式細胞儀進行檢測,結果發(fā)現與對照組相比,JNK抑制劑作用后接毒組細胞凋亡率明顯上升,差異極顯著(P0.01),此時ATF3表達量降低,說明ATF3在細胞內起到抑制凋亡的作用;同時,與對照組相比,JNK抑制劑作用后接毒組細胞周期發(fā)生阻滯。N2a細胞感染PHEV后,ATF3的表達量增高。JNK抑制劑作用于N2a細胞后,ATF3表達量下降,細胞凋亡率上升,細胞周期阻滯,DNA合成受阻,細胞活力下降,抗病毒能力下降。表明神經細胞感染PHEV后JNK信號通路可調控ATF3的表達,進而影響神經細胞凋亡和細胞周期。
[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.
【學位授予單位】：沈陽農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.65
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本文編號：1632727