小反刍兽疫病毒N75-1株入侵宿主细胞的途径研究

发布时间：2018-04-16 09:34

本文选题：小反刍兽疫病毒 + 山羊子宫内膜上皮细胞　；参考：《西北农林科技大学》2017年硕士论文

【摘要】：小反刍兽疫(Peste des petits ruminants,PPR)是由小反刍兽疫病毒(Peste des petits ruminants virus,PPRV)引起的感染小反刍兽的一类动物传染病。近年来,PPRV的研究大多集中在蛋白功能以及疫苗制备等方面,有关PPRV入侵宿主细胞的途径研究少有报道。本试验目的在于探究PPRV对山羊子宫内膜上皮细胞(Goat endometrial epithelial cells,EEC)的感染特性,以及病毒进入宿主细胞的内吞途径,从而为研究该病毒对宿主细胞的感染机制提供相关的理论基础及科学依据。研究结果如下:1.获得了PPRV在山羊子宫内膜上皮细胞上的增殖规律。PPRV接种于EEC细胞后24 h到48 h后细胞无明显的形态学变化,72h发现细胞发生典型的细胞病变(cytopathic effect,CPE),细胞聚集在一起,形成合胞体,折光性变强等,96 h后细胞出现裂解、死亡现象。EEC接种PPRV N75-1株后,激光共聚焦观察确定该病毒进入细胞的时间在1-2 h内。收取1 h、3 h、6 h、9 h、12 h、24 h的细胞上清,TCID50检测发现病毒滴度在感染后24 h达到最高;收取0 h、3 h、6 h、9 h、12 h、24 h的细胞样品,Western Blot检测表明N蛋白的表达在感染后12 h表达量最大,24h有所下降,获得了PPRV在EEC细胞上的增殖规律。2.PPRV入侵EEC不通过网格蛋白介导的内吞途径。用内吞途径的网格蛋白抑制剂氯丙嗪(CPZ)处理EEC,接毒后激光共聚焦显微镜观察进入细胞的PPRV与对照组相比无明显差异;Western Blot以及TCID50检测发现N蛋白的表达和毒价亦无明显变化。将网格蛋白的的siRNA转染EEC中,接毒后Western Blot以及TCID50检测N蛋白的表达以及PPRV毒价亦无明显变化。实验结果表明,PPRV入侵EEC不通过网格蛋白介导的内吞途径。3.PPRV入侵EEC是通过Caveola介导的Qg吞途径。用膜穴样凹陷内吞途径抑制剂甲基-β-环糊精(MβCD)和制霉素(Nystatin)两种药物处理EEC,接种PPRV后激光共聚焦显微镜观察到进入细胞的病毒相对于对照组明显减少。Western Blot检测发现,药物处理组PPRV-N蛋白表达与对照相比均显著减少。将Caveolin-1的siRNA转染EEC后接种PPRV,Western Blot以及TCID50检测发现N蛋白表达和病毒滴度明显降低。实验结果表明,PPRV入侵EEC是通过Caveola介导的Qg吞途径。综上所述,本研究通过特异性化学抑制剂阻断、siRNA干扰方法确定,PPRV入侵EEC是依赖膜穴样凹陷介导的内吞途径。研究结果为了解PPRV的致病机制和寻找新的药物治疗靶点提供理论依据。
[Abstract]:Peste des petits ruminants (PPRV) is a kind of infectious disease of small ruminants caused by Peste des petits ruminants virus (PPRV).In recent years, the research of PPRV is mainly focused on protein function and vaccine preparation. There are few reports on the pathway of PPRV invading host cells.The purpose of this study was to investigate the characteristics of PPRV infection on goat endometrial epithelial cells (Goat endometrial epithelial cells) and the endocytosis pathway of the virus into host cells.Therefore, it provides a theoretical and scientific basis for the study of the infection mechanism of the virus on host cells.The results are as follows: 1.The rule of proliferation of PPRV in goat endometrial epithelial cells was obtained. There were no obvious morphological changes in EEC cells after inoculation with PPRV for 24 h to 48 h. The typical cytopathic effect PPRV cells were found to have a typical cytopathic effect PPRV, and the cells gathered together to form syncytosomes.After 96 h of refractive change, the cells were lysed. The death phenomenon. After inoculating PPRV N75-1 strain, the laser confocal observation confirmed that the time of the virus entering the cells was within 1-2 hours.TCID50 titer of cell supernatant was detected at 24 h after infection, and the highest titer of TCID50 was found at 24 h after infection, and the expression of N protein was decreased at 12 h after infection by Western Blot analysis of cell samples collected for 0 h ~ 3 h ~ 6 h ~ 9 h ~ 9 h ~ 12 h ~ 24 h.The proliferation of PPRV on EEC cells was obtained. 2. PPRV invades EEC through endocytosis without grid protein.There was no significant difference in the expression of N protein and the toxic value between the PPRV cells and the control group by laser confocal microscopy after treatment with chlorpromazine (CPZ), a grid protein inhibitor of endocytosis pathway. The expression of N protein and the toxic value of N protein were also found to be unchanged by TCID50.The siRNA of griddle protein was transfected into EEC. The expression of N protein and PPRV level were not significantly changed by Western Blot and TCID50.The results showed that the invasion of EEC by PPRV was not mediated by the endocytosis pathway mediated by grid protein. 3. The invasion of EEC by PPRV was mediated by Caveola.EECs were treated with two drugs, methyl- 尾 -cyclodextrin (M 尾 CDD) and Nystatin (2). After inoculation with PPRV, the number of viruses entering the cells was significantly decreased compared with the control group. Western Blot analysis showed that the number of viruses entering the cells was significantly lower than that in the control group.The expression of PPRV-N protein in the drug treated group was significantly lower than that in the control group.The expression of N protein and the titer of the virus were significantly decreased after the siRNA of Caveolin-1 was transfected into EEC, and the expression of N protein and the titer of the virus were detected by TCID50 assay.The results showed that PPRV invasion of EEC was mediated by Caveola-mediated QG-swallowing pathway.To sum up, the method of blocking siRNA interference by specific chemical inhibitors confirmed that PPRV invasion of EEC was a membrane-like hole-mediated endocytosis pathway.The results provide theoretical basis for understanding the pathogenesis of PPRV and finding new therapeutic targets.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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