人类肠道病毒68型干扰宿主细胞周期以促进自身病毒复制的机制研究

发布时间：2018-01-11 00:22

本文关键词：人类肠道病毒68型干扰宿主细胞周期以促进自身病毒复制的机制研究　出处：《吉林大学》2017年博士论文　论文类型：学位论文

【摘要】：背景:人类肠道病毒68型(EV-D68)可在儿童中引起严重的呼吸系统疾病,且在某些严重病例中可发生急性弛缓性瘫痪及颅脑神经功能障碍等严重的神经系统并发症,是一种可致命的病原体。EV-D68首次发现于1962年,是从加利福尼亚州的4名患有肺炎及支气管炎的儿童体内分离提取出的。在过去的10年里,意大利、美国、德国、中国及几个其他的国家均有EV-D68感染爆发的报道。且在2014年,在美国和加拿大爆发了截至目前为止规模最大,波及范围最广泛且伴随着严重的临床表现的EV-D68感染。不幸的是,针对EV-D68感染目前没有特效的预防用的疫苗及治疗用的药物。这主要是由于截至目前为止,我们对于EV-D68复制所需要的宿主细胞的细胞因子所知甚少。众所周期,许多病毒是通过调节宿主细胞的细胞周期进程来促进自身的复制,这是它们致病机制中的一个特征。在之前的研究中,我们发现人类肠道病毒71型(EV-A71)和柯萨奇病毒A16(CA16)都可以引起宿主细胞S期阻滞以促进其本身的病毒复制。然而EV-D68是否存在潜在的细胞周期操控能力,且其对细胞周期的操控能力是否与其最近爆发时的强致病性及致死性有关等问题到目前为止都没有被研究。目的:在本次研究中,我们针对不同细胞周期状态对EV-D68复制的影响及EV-D68对宿主细胞周期的操控能力进行了探讨。以求进一步增加对EV-D68致病机理的理解,并且尽力为日后EV-D68感染相关疾病的预防及特异性抗病毒治疗提供潜在的靶点。方法:为了研究不同细胞周期状态对EV-D68复制的影响及EV-D68病毒对宿主细胞的细胞周期方面的影响,我们以无血清的DMEM培养RD细胞使细胞处于G0/G1期同步化状态,以0.85 m M的胸腺嘧啶核苷处理RD细胞使其处于S期同步化状态,且以25 ng/ml的诺考达唑处理细胞使其处于G2/M期同步化状态。在实验过程中,我们通过流式细胞术来测定宿主细胞的细胞周期曲线,且通过Mod Fit LT软件来分析处于不同细胞周期阶段的细胞占总细胞数的百分比。在基因水平,我们通过实时定量PCR技术测定RD细胞内EV-D68的RNA水平及各种细胞周期相关蛋白的m RNA表达水平。为了测定病毒的毒力,我们以滴定法测定上清液中的子代病毒。我们通过半定量的蛋白免疫印迹方法(western blot)对EV-D68感染后细胞周期相关蛋白的调节进行了分析,且利用Elisa试剂盒对各种细胞周期相关蛋白的表达量进行了测定以验证蛋白免疫印迹所得结果的正确性。结果:实验结果表明,同步化宿主细胞周期到G0/G1期可以促进EV-D68扩增,S期同步化没有促进病毒扩增的能力,但G2/M期同步化可以抑制病毒的复制。无论是最早被分离的EV-D68的Fermon株,还是目前正在全球范围内流行的EV-D68的几株流行株都可以调控宿主细胞的细胞周期使其发生G0/G1期阻滞,进而为EV-D68病毒的复制提供最适宜的环境。从蛋白质表达水平和/或相应基因的m RNA的表达水平上可以发现,EV-D68对细胞周期的调节与细胞周期蛋白及细胞周期蛋白依赖性激酶表达的相关作用有关。此外,我们还发现,EV-D68的病毒非结构蛋白3D可以阻止细胞周期由G0/G1期进入S期。有趣的是,EV-D68与EV-A71虽同为肠道病毒,但G0/G1期同步化并不能促进EV-A71的复制,相反G0/G1期同步化却抑制EV-A71的复制。然而,这两种病毒却存在一个相似点:G2/M期同步化对这两种病毒的复制及活性均起到抑制作用,这为肠道病毒的常规治疗提供了一个靶点和方向。这些结果进一步的解释了肠道病毒特别是EV-D68的致病机制,且为日后EV-D68相关疾病的预防和治疗提供了潜在的策略。结论:不同于对EV-A71的研究,本次研究的结果表明EV-D68显示出了增加宿主细胞中G0/G1期细胞所占百分比的显著能力,且G0/G1期是EV-D68进行复制的最适宜的环境。且我们还发现G2/M期阻滞既可以抑制EV-D68的复制也可以抑制EV-A71的复制。因此,我们推测诱导G2/M阻滞的药物可以被认为是抑制不同类型的肠道病毒感染的通用抗病毒疗法。这为将来抗肠道病毒及抗EV-D68药物的发展提供了新的方向。
[Abstract]:Background: human enterovirus 68 (EV-D68) can cause severe respiratory disease in children, and in some severe cases can be acute flaccid paralysis and cranial nerve dysfunction and other serious neurological complications, is a deadly pathogen.EV-D68 was first discovered in 1962, is to extract isolated from 4 patients suffering from pneumonia and bronchitis in children in California. In the past 10 years, Italy, the United States, Germany, Chinese and several other countries have reported outbreaks of EV-D68 infection. And in 2014, in the United States and Canada broke out so far the largest and most widely spread and accompanied by clinical severe EV-D68 infection. Unfortunately, vaccines and drugs for the treatment of EV-D68 infection prevention and there is no specific use. This is mainly because so far, we have to The cytokines are required for EV-D68 replication in host cells is poorly understood. The cycle of many viruses is to promote their own replication through the process of cell cycle regulation of host cells, which is a feature of their pathogenesis. In previous studies, we found that human enterovirus 71 (EV-A71) and Coxsackie virus A16 (CA16) can cause the host cell S phase arrest to promote viral replication itself. However, the existence of EV-D68 cell cycle control potential, and the ability to control the cell cycle and the recent outbreak of the highly pathogenic and lethal related problems have not been studied so far Objective: in this study, we focused on the different cell cycle effect on replication of EV-D68 and EV-D68 on the cell cycle control ability are discussed. In order to further increase of EV-D68 The mechanism of disease understanding, and try to provide potential targets for diseases related to EV-D68 infection after prevention and specific antiviral therapy. Methods: To study the effects of different cell cycle effects on EV-D68 replication and EV-D68 virus to host cells of the cell cycle, we cultured RD cells in serum-free DMEM cell in G0/G1 phase synchronization state, with 0.85 m M thymidine treatment of RD cells in the S phase synchronization state, and in 25 ng/ml nocodazole treated cells which in G2/M phase synchronization state. During the experiment, cell cycle curve by flow cytometry to determine the host cell and through the Mod Fit software to analyze LT in different stages of the cell cycle cell percentage of the total cell population. At the genetic level, we measured RD EV-D68 cells by real-time quantitative PCR technology RN The expression level of M RNA A and the level of various cell cycle related proteins. In order to virulence of the virus, we measured in the supernatants of progeny virus by titration. We through Western blot semi quantitative method (Western blot) on the regulation of cell cycle related proteins after EV-D68 infection were analyzed, and expression by Elisa kit for various cell cycle related proteins were measured to verify the correctness of the results of the Western blot. Results: the experimental results show that the synchronization of host cells into G0/G1 phase can promote EV-D68 amplification, S phase synchronization did not promote the ability of the virus amplification, but G2/M phase synchronization can inhibit the replication of the virus. It is the first isolated EV-D68 strains of Fermon, and is currently a pandemic of EV-D68 few epidemic strains can cell cycle regulation of host cells to make it G0/G1 arrest, provide the most suitable environment for EV-D68 replication. And that the level of M expression level of RNA and / or the corresponding gene from protein expression can EV-D68 on cell cycle regulation role and cyclin and cyclin dependent kinases. In addition, we also found that EV-D68, the virus nonstructural protein 3D can prevent the cell cycle from G0/G1 phase to S phase. Interestingly, EV-D68 and EV-A71 belong to the intestinal virus, but G0/G1 phase synchronization does not promote EV-A71 replication, whereas G0/G1 phase synchronization is to inhibit the replication of EV-A71. However, there is a similarity between the two virus: G2/M phase synchronization and replication activity of the two kinds of viruses play inhibition, provides a target and direction of the routine treatment of intestinal viruses. These results further explain the intestinal tract Virus especially the pathogenic mechanism of EV-D68, provides a potential strategy for the prevention and treatment of EV-D68 related diseases. Conclusion: different from the day after the study of EV-A71, the results of this study indicated that EV-D68 showed significant increase in capacity of G0/G1 phase cells in the host cell percentage, and G0/G1 is EV-D68 copy of the most appropriate environment. And we also found that G2/M arrest can inhibit the replication of EV-D68 can inhibit the replication of EV-A71. Therefore, we speculate that drug induced G2/M block can be considered universal antiviral therapy inhibits intestinal virus of different types of infection. This provides a new direction for the future development of anti intestinal virus and anti EV-D68 drugs.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R373.2

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