HD-11细胞感染H9N2流感病毒后先天性免疫相关基因mRNA表达变化的研究

发布时间：2018-11-17 12:44

【摘要】：H9N2亚型禽流感病毒(H9N2 avian influenza virus,AIV)是低致病性的流感病毒。鸡群感染流感病毒后引起免疫系统功能抑制,对多种病原易感性增加,易继发感染大肠杆菌等细菌性疾病或更严重的临床疾病。HD-11是鸡巨噬细胞,是禽重要的先天免疫细胞。本研究通过H9N2 AIV感染HD-11细胞模拟机体抗病毒感染的先天免疫反应,利用免疫荧光方法特异的检测H9N2流感病毒在细胞内的感染情况;用1 MOI H9N2 AIV感染HD-11细胞,收集感染后0,6,12,24,36,48,72 h的细胞样品,利用实时荧光定量PCR技术检测细胞模式识别受体,细胞因子,趋化因子以及iNOS基因mRNA表达变化量。通过上述研究方法,我们取得了如下试验结果:1.HD-11细胞感染病毒12 h后,椭圆形或梭形的HD-11细胞开始逐渐皱缩变圆,随后出现胞膜边缘不齐、拉丝、聚团、空泡、脱落等明显的细胞病变现象。感染H9N2AIV 36 h后,H9N2 AIV NP蛋白单抗免疫荧光染色,细胞核周围可见明亮绿色,流感病毒在细胞内分布。2.实时荧光相对定量结果显示chTLR7 mRNA在感染后12 h开始显著上调,感染后48 h,上调3.8倍,达到顶峰。chMDA5 mRNA在感染后一直显著上调,72 h上调到102.5倍。chMDA5 mRNA上调作用较chTLR7mRNA更强些。IL-1β,IL-2,IL-6细胞因子mRNA随着流感病毒作用时间延长开始显著上调,36h表达量上调到最大值,随后呈显著下调趋势但仍高于对照组。IFN-α,IFN-βI型干扰素的mRNA转录水平从感染后24 h开始显著上调,72 h分别上调到48.8倍和11.3倍。XCL1和CXCLi1趋化因子mRNA在感染后12 h开始显著上调,XCL1 mRNA 36h上调值最大,CXCLi1趋化因子mRNA在感染后48 h达到最大值,然后都开始下调;CXCLi2趋化因子mRNA在感染后36 h开始显著上调,72 h上调到20倍。iNOS mRNA在病毒感染后12 h开始显著上调,36 h上调到最大值,上调4.3倍。初步探索了流感病毒对机体先天免疫功能的影响。
[Abstract]:H9N2 subtype avian influenza virus (H9N2 avian influenza virus,AIV) is a low-pathogenic influenza virus. Chickens infected with influenza virus cause inhibition of immune system, increase susceptibility to various pathogens, and secondary infection with bacterial diseases such as Escherichia coli or more serious clinical diseases. HD-11 is a chicken macrophage. It is an important innate immune cell in birds. In this study, HD-11 cells infected with H9N2 AIV were used to simulate the innate immune response of human body to antiviral infection, and specific immunofluorescence method was used to detect the infection of H9N2 influenza virus in cells. HD-11 cells were infected with 1 MOI H9N2 AIV. The cell samples were collected for 72 h after infection. The expression of cell pattern recognition receptor, cytokines, chemokines and iNOS gene mRNA were detected by real-time fluorescence quantitative PCR. Through the above research methods, we obtained the following results: after 1.HD-11 cells were infected with the virus for 12 h, the elliptical or fusiform HD-11 cells began to shrink and round gradually, and then the membrane edge was irregular, drawing wire, agglomeration, vacuoles, and so on. Abscission and other obvious cytopathic phenomena. After 36 h of H9N2AIV infection, the H9N2 AIV NP protein monoclonal antibody was stained with immunofluorescence, the nucleus around the nucleus was bright green, and the influenza virus was distributed in the cells. The results of real-time fluorescence relative quantitative analysis showed that chTLR7 mRNA increased significantly at 12 h after infection and 3.8 times at 48 h after infection, reaching its peak. ChMDA5 mRNA was significantly up-regulated after infection. The up-regulation of chMDA5 mRNA was stronger than that of chTLR7mRNA at 72 h. IL-1 尾, IL-2,IL-6 cytokine mRNA began to be upregulated significantly with the prolongation of influenza virus exposure, and the expression of mRNA increased to the maximum at 36h. The mRNA transcription level of IFN- 伪, IFN- 尾 I interferon increased significantly 24 hours after infection. XCL1 and CXCLi1 chemokine mRNA began to increase significantly at 12 h after infection, XCL1 mRNA at 36 h was the highest, and CXCLi1 chemokine mRNA reached the maximum at 48 h after infection, and then down-regulated. CXCLi2 chemokine mRNA increased significantly at 36 h after infection, increased to 20 fold at 72 h, increased significantly at 12 h after infection, reached the maximum at 36 h, and increased 4.3 times. The effect of influenza virus on innate immune function was preliminarily explored.
【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

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