自噬抑制剂减少禽呼肠孤病毒诱导的细胞及鸡胚组织凋亡

发布时间：2018-02-11 10:36

  本文关键词： 禽呼肠孤病毒 自噬抑制剂 CQ E64d+pepstatinA 凋亡　出处：《扬州大学》2015年硕士论文　论文类型：学位论文

【摘要】：禽呼肠孤病毒(avian reovirus, ARV)感染可引起鸡和火鸡的病毒性关节炎、腱鞘炎和免疫抑制,给养禽业造成重大损失。已证实ARV感染能诱导细胞自噬和凋亡,且与病毒的致病作用有关,但如何降低ARV诱导的细胞凋亡有待进一步研究。本研究旨在探索自噬抑制剂CQ及E64d+pepstatinA对ARV诱导细胞凋亡的影响。首先使用MTT法检测两种自噬抑制剂对ARV诱导的细胞生长抑制的影响。结果显示,经两种自噬抑制剂预处理的DF1细胞感染ARV后,病毒诱导的细胞生长抑制率显著降低。进一步经形态学观察发现,两种自噬抑制剂作用后,ARV诱导的DF1细胞融合病变显著减少。最后,使用PI和Annexin V-FITC双染法进行流式细胞术检测,结果显示,经CQ或E64d+pepstatinA处理后,ARV诱导的DFl细胞凋亡率显著降低。Caspase-3的裂解水平高低通常被认为是凋亡发生强弱的标志。本实验采用western blot法检测ARV (GX/2010/1株)感染的DF1细胞caspase-3活性,发现经CQ或E64d+pepstatinA预处理后,ARV诱导的DFl细胞凋亡率显著降低。进一步进行病毒衣壳蛋白σ℃表达检测及病毒滴度测定,发现两种自噬抑制剂均可显著降低病毒复制水平。禽呼肠孤病毒可利用凋亡使感染禽产生病理反应,鉴于两种自噬抑制剂可在细胞水平抑制ARV诱导的细胞凋亡,进一步进行了动物实验。使用CQ或E64d+pepstatinA对11日龄鸡胚预处理后,接种ARV病毒(GX/2010/1株),并设立对照组。收集尿囊液进行病毒滴度测定,发现经两种自噬抑制剂处理后,病毒滴度显著低于单独接毒组。收集组织样品进行蛋白检测,并统计鸡胚死亡率,绘制生存率曲线。结果显示,CQ或E64d+pepstatinA均可显著抑制ARV诱导的鸡胚法氏囊、心脏及肠组织细胞凋亡,进一步降低鸡胚死亡率。本研究证明自噬抑制剂CQ和E64d+pepstatinA在抑制自噬的基础上可显著抑制ARV(GX/2010/1株)诱导的细胞凋亡,降低病毒复制水平；对鸡胚,可显著降低ARV(GX/2010/1株)导致的胚体死亡率。
[Abstract]:Avian reovirus virus (ARV) infection can cause viral arthritis, tenosynovitis and immunosuppression in chickens and turkeys. It has been proved that ARV infection can induce autophagy and apoptosis, and is related to the pathogenicity of the virus. However, how to reduce apoptosis induced by ARV needs further study. The purpose of this study is to explore the effect of autophagy inhibitor CQ and E64d pepstatinA on apoptosis induced by ARV. Firstly, MTT assay was used to detect the effect of two kinds of autophagy inhibitors on ARV induced apoptosis. The effect of cell growth inhibition. After two kinds of autophagy inhibitors pretreated DF1 cells infected with ARV, the inhibition rate of cell growth induced by virus was significantly decreased. After treatment with two autophagy inhibitors, the DF1 cell fusion lesion induced by ARV was significantly reduced. Finally, flow cytometry was performed with Pi and Annexin V-FITC staining, and the results showed that, The apoptosis rate of DFl cells induced by CQ or E64d pepstatinA was significantly decreased. The level of cleavage of Caspase-3 was generally regarded as a marker of the degree of apoptosis. The caspase-3 activity of DF1 cells infected with ARV GX / 2010 / 1 strain was detected by western blot assay. It was found that the apoptosis rate of DFl cells induced by CQ or E64d pepstatinA was significantly decreased, and the expression of virus capsid protein 蟽 鈩，

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