A549-NS1稳转株中NS1-NOLC1相互作用对CK2表达水平、细胞增殖、病毒复制影响的初步研究

发布时间：2018-04-01 08:29

本文选题：NS1蛋白　切入点：NOLC1蛋白　出处：《辽宁大学》2017年硕士论文

【摘要】：A型流感病毒属正粘病毒科,为单股负链RNA病毒。至今,世界性的大规模流感疫情的爆发都是由A型流感病毒引起的,并且新型的禽流感病毒已经变得更容易在人与人之间传播,因此禽流感病毒的传染性和致病性必须引起广泛关注。A型流感病毒的NS1蛋白(nonstructural protein 1)仅存在于被感染的细胞中,在细胞中发挥着抑制宿主pre-mRNA剪接、拮抗干扰素、促进病毒复制等重要功能。人核仁螺旋体磷酸化蛋白(NOLC1)是一个高度磷酸化的哺乳动物蛋白,既参与核仁的发生和rDNA的转录,又参与炎症发生、细胞生长、细胞凋亡、肿瘤发生等相关信号通路的调控。在本实验中我们主要研究NS1蛋白与NOLC1蛋白相互作用对宿主细胞增殖、凋亡和病毒复制的影响及其相关信号通路。本研究用慢病毒转染方法构建NS1 A549稳定转染细胞株,免疫荧光和Western Blot检测稳转株NS1蛋白的表达,结果显示稳转细胞株构建成功。为了深入研究NS1与NOLC1相互作用对细胞凋亡的影响,本研究采用ELISA的方法确定了A549、A549-FLAG、A549-FLAG-NS1细胞中的CK2的表达水平,采用Real-time PCR和Western Blot的方法检测了A549、A549-FLAG、A549-FLAG-NS1细胞中NOLC1和NF-κB的mRNA及蛋白水平,结果显示NS1与NOLC1相互作用降低了CK2的表达水平,上调了NOLC1的mRNA水平,抑制了NOLC1的蛋白表达,同时提高了细胞中NF-κB的mRNA水平,有文献报道CK2、NOLC1和NF-κB是调控细胞凋亡的关键因子,结合本实验室之前的研究结果,我们推测NS1与NOLC1相互作用促进细胞凋亡与其调控CK2、NOLC1和NF-κB的表达水平相关,具体的机制还有待于进一步研究。为检测NS1与NOLC1相互作用相互作用对病毒复制能力的影响,本实验用H3N2禽流感病毒感染A549、A549-FLAG、A549-FLAG-NS1细胞,用Real-time PCR检测宿主细胞中病毒NP三种RNA水平来反映病毒复制情况,结果显示NS1与NOLC1相互作用促进了病毒的复制。为检测NS1与NOLC1相互作用对宿主细胞增殖能力的影响,本实验用MTT法对A549、A549-FLAG、A549-FLAG-NS1细胞株的增殖能力进行检测,结果显示NS1与NOLC1相互作用提高了细胞的增殖能力。通过上述研究,我们确定了NS1与NOLC1相互作用影响宿主细胞的增殖和凋亡,为进一步研究相关的信号通路,本研究采用Real-time PCR技术对A549、A549-FLAG、A549-FLAG-NS1中与细胞增殖和凋亡相关的多个基因进行了检测,结果显示其中Wnt信号通路中的β-catenin和cyclinD基因、Bcl2和CDK4基因与两种蛋白的相互作用相关,NS1与NOLC1相互作用上调了β-catenin、cyclinD和Bcl2的mRNA水平,对CDK4的mRNA水平起下调作用,NS1可能通过调控这些基因及其信号通路来调控细胞的增殖和凋亡。研究NS1与NOLC1相互作用对宿主细胞增殖、凋亡和病毒复制的影响及相关信号通路,有助于我们深入研究NS1与NOLC1相互作用的机制,为禽流感致病机理的研究提供了新的思路。
[Abstract]:Influenza A virus belongs to Orthomyxoviridae, which is a single negative strand RNA virus.So far, worldwide outbreaks of large-scale influenza have been caused by influenza A viruses, and new avian influenza viruses have become more easily transmitted from person to person.Therefore, the infectivity and pathogenicity of avian influenza virus (Avian Influenza virus) must attract wide attention. The nonstructural protein 1 (NS1 protein) of influenza virus type A only exists in infected cells. It plays an important role in inhibiting pre-mRNA splicing and antagonizing interferon in the cells.Promote virus replication and other important functions.Human nucleolar spirochete phosphorylated protein (NOLC1) is a highly phosphorylated mammalian protein involved in the regulation of signal pathways such as nucleolus formation and rDNA transcription, inflammation, cell growth, apoptosis, tumorigenesis, and so on.In this study, we studied the effects of interaction between NS1 protein and NOLC1 protein on host cell proliferation, apoptosis and virus replication, and related signaling pathways.In this study, NS1 A549 stable transfection cell line was constructed by lentivirus transfection. The expression of NS1 protein was detected by immunofluorescence and Western Blot. The results showed that the stable transformed cell line was successfully constructed.In order to study the effect of NS1 and NOLC1 interaction on apoptosis, the expression of CK2 in A549A549-FLAG-NS1 cells was determined by ELISA. The mRNA and protein levels of NOLC1 and NF- 魏 B in A549-FLAG-NS1 cells were detected by Real-time PCR and Western Blot.The results showed that the interaction of NS1 and NOLC1 decreased the expression of CK2, up-regulated the mRNA level of NOLC1, inhibited the expression of NOLC1 protein, and increased the mRNA level of NF- 魏 B. CK2NOLC1 and NF- 魏 B were reported to be the key factors in regulating apoptosis.Based on our previous results, we speculate that the interaction between NS1 and NOLC1 promotes apoptosis related to the regulation of CK2NOLC1 and NF- 魏 B expression, and the specific mechanism remains to be further studied.In order to detect the effect of interaction between NS1 and NOLC1 on viral replication, we used H3N2 avian influenza virus to infect A549 FLAGN A549-FLAG-NS1 cell line, and Real-time PCR was used to detect the level of NP three kinds of RNA in host cells to reflect the viral replication.The results showed that the interaction of NS1 and NOLC1 promoted the replication of the virus.In order to detect the effect of NS1 and NOLC1 interaction on the proliferation of host cells, the proliferation ability of A549A549-FLAG-NS1 cell line was detected by MTT method. The results showed that the interaction between NS1 and NOLC1 enhanced the proliferation of A549- FLAG-NS1 cell line.Through the above studies, we have determined that the interaction between NS1 and NOLC1 affects the proliferation and apoptosis of host cells.In this study, Real-time PCR technique was used to detect several genes related to cell proliferation and apoptosis in A549A549-FLAG-NS1, A549-FLAG-NS1, A549-FLAG-NS1 and A549-FLAG-NS1.The results showed that 尾 -catenin and cyclinD genes Bcl2 and CDK4 genes in the Wnt signaling pathway were related to the interaction between the two proteins. The interaction between NS1 and NOLC1 up-regulated the mRNA levels of 尾 -catenin D and Bcl2.NS1 may regulate the proliferation and apoptosis of CDK4 cells by regulating these genes and their signaling pathways.The study of the effect of NS1 / NOLC1 interaction on host cell proliferation, apoptosis and virus replication and related signaling pathways is helpful for us to study the mechanism of NS1 / NOLC1 interaction and provide a new idea for the study of the pathogenesis of avian influenza.
【学位授予单位】：辽宁大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R373

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