H9N2禽流感病毒NS1蛋白诱导的氧化应激促进DF-1细胞凋亡

发布时间：2018-07-04 19:19

本文选题：H9N2亚型禽流感病毒 + NS1蛋白　；参考：《西北农林科技大学》2015年硕士论文

【摘要】：H9N2禽流感NS1蛋白是该病毒的非结构蛋白,而且是一种多功能调节蛋白,能够抵制流感病毒感染所引发的宿主免疫反应,而且还可以导致多种传代细胞凋亡。在我们之前的研究中已经证实,H9N2禽流感病毒NS1蛋白能够诱导DF-1细胞发生凋亡。为了研究H9N2禽流感病毒NS1蛋白是否能够引起氧化应激以及氧化应激在其致凋亡作用中的影响,我们将质粒pEGFP-NS1转染DF-1细胞不同时间后,检测细胞内活性氧的累积情况及氧化还原信号的变化;将pEGFP-NS1转染DF-1细胞48小时,并设置空白对照组、空载体对照组以及抗氧化剂(PTDC和NAC)处理组,探索氧化应激对细胞凋亡的影响。研究结果如下:1.H9N2禽流感病毒NS1蛋白能够引起DF-1细胞产生氧化应激。我们于荧光显微镜下观察活性氧阴离子探针红色荧光发现,在pEGFP-NS1转染24小时后ROS的产生开始增多,并随时间延长而增加,通过流式细胞术定量检测进一步证实这一结果;超氧化物歧化酶和过氧化氢酶活性的检测结果表明,两者的活性都随着pEGFP-NS1转染时间的延长升高;相对荧光定量PCR检测PRDX3和TRX的mRNA表达水平结果显示,pEGFP-NS1转染24小时开始,两者均有不同程度的下调。2.氧化应激促进H9N2禽流感病毒NS1蛋白诱导的DF-1细胞凋亡过程。本实验设置空白对照组、空载体对照组、pEGFP-NS1转染48小时、PTDC/NAC预处理的pEGFP-NS1转染组共5组细胞。经流式细胞术检测,各组凋亡率为:2.4%、4.0%、15.8%、12.8%、6.8%,抗氧化剂处理组细胞凋亡率明显低于实验组;caspase-3、caspase-8、caspase-9活性检测结果,抗氧化剂处理组细胞的caspase-3、caspase-9活性均降低,caspase-8活性变化不明显;Western Blotting检测活化的caspase-3、caspase-8、caspase-9、Bax及bcl-2的蛋白表达情况发现,与caspases活性检测结果相一致,抗氧化剂处理后活化的caspase-3、caspase-9的表达量降低,caspase-8表达量变化不明显,抗氧化剂处理后Bax的表达比实验组低,而bcl-2则比实验组要高。综上所述,本实验研究证实了H9N2禽流感病毒NS1蛋白能够诱导DF-1细胞产生氧化应激,且氧化应激参与其诱导的DF-1凋亡过程。且氧化应激可能通过内源性凋亡路径(线粒体依赖性凋亡途径)对H9N2禽流感病毒NS1蛋白诱导的DF-1细胞凋亡过程进行调节。
[Abstract]:H9N2 avian influenza NS1 protein is a non-structural protein of the virus and a multifunctional regulatory protein which can resist the host immune response caused by influenza virus infection and can induce apoptosis of many passage cells. Our previous studies have confirmed that H9N2 avian influenza virus NS1 protein can induce apoptosis in DF-1 cells. In order to study whether NS1 protein of H9N2 avian influenza virus can induce oxidative stress and the effect of oxidative stress on apoptosis, we transfected the plasmid pEGFP-NS1 into DF-1 cells for different time. After transfection of pEGFP-NS1 into DF-1 cells for 48 hours, blank control group, empty carrier control group and antioxidant treatment group (PTDC and NAC) were set up. To explore the effect of oxidative stress on apoptosis. The results are as follows: 1. The NS1 protein of H9N2 avian influenza virus can induce oxidative stress in DF-1 cells. We observed the red fluorescence of reactive oxygen species anion probe under fluorescence microscope and found that Ros production began to increase after 24 hours of pEGFP-NS1 transfection and increased with time. This result was further confirmed by flow cytometry. The activity of superoxide dismutase and catalase increased with the prolongation of pEGFP-NS1 transfection time, and the mRNA expression level of PRDX3 and TRX by relative fluorescence quantitative PCR showed that pEGFP-NS1 transfection began 24 hours after transfection. Both have varying degrees of down-regulation. Oxidative stress promotes apoptosis of DF-1 cells induced by NS1 protein of H9N2 avian influenza virus. In this experiment, blank control group and empty vector control group pEGFP-NS1 were transfected with pEGFP-NS1 pretreated with PTDC / NAC for 48 hours. By flow cytometry, the apoptotic rate of each group was 1: 2. 4%. The apoptosis rate of the antioxidant treated group was significantly lower than the activity of caspase-3, caspase-8, caspase-8 and caspase-9 in the antioxidant treatment group, and the apoptotic rate was 12. 8% and 12. 8% respectively, and the apoptosis rate of the antioxidant treatment group was significantly lower than that of the experimental group. The activities of caspase-3, caspase-9 and caspase-9 in antioxidant treated cells were not significantly changed. Western blotting was used to detect the expression of caspase-3, caspase-8, caspase-9, Bax and bcl-2, which was consistent with the results of caspases activity test. The expression of activated caspase-3 and caspase-9 after antioxidant treatment had no significant change. The expression of Bax was lower than that of experimental group, but the expression of bcl-2 was higher than that of experimental group. In conclusion, this study confirmed that NS1 protein of H9N2 avian influenza virus can induce oxidative stress in DF-1 cells, and oxidative stress participates in the process of DF-1 apoptosis induced by H9N2 avian influenza virus. Oxidative stress may regulate the apoptosis of DF-1 cells induced by NS1 protein of H9N2 avian influenza virus through endogenous apoptosis pathway (mitochondrial dependent apoptosis pathway).
【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】：2015
【分类号】：S852.65

【相似文献】