發(fā)布時間：2018-01-11 00:22

  本文關(guān)鍵詞：人類腸道病毒68型干擾宿主細(xì)胞周期以促進(jìn)自身病毒復(fù)制的機(jī)制研究　出處：《吉林大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 人類腸道病毒68型(EV-D68) 細(xì)胞周期 G0/G1期阻滯 病毒復(fù)制 宿主-病原體相互作用

【摘要】：背景:人類腸道病毒68型(EV-D68)可在兒童中引起嚴(yán)重的呼吸系統(tǒng)疾病,且在某些嚴(yán)重病例中可發(fā)生急性弛緩性癱瘓及顱腦神經(jīng)功能障礙等嚴(yán)重的神經(jīng)系統(tǒng)并發(fā)癥,是一種可致命的病原體。EV-D68首次發(fā)現(xiàn)于1962年,是從加利福尼亞州的4名患有肺炎及支氣管炎的兒童體內(nèi)分離提取出的。在過去的10年里,意大利、美國、德國、中國及幾個其他的國家均有EV-D68感染爆發(fā)的報道。且在2014年,在美國和加拿大爆發(fā)了截至目前為止規(guī)模最大,波及范圍最廣泛且伴隨著嚴(yán)重的臨床表現(xiàn)的EV-D68感染。不幸的是,針對EV-D68感染目前沒有特效的預(yù)防用的疫苗及治療用的藥物。這主要是由于截至目前為止,我們對于EV-D68復(fù)制所需要的宿主細(xì)胞的細(xì)胞因子所知甚少。眾所周期,許多病毒是通過調(diào)節(jié)宿主細(xì)胞的細(xì)胞周期進(jìn)程來促進(jìn)自身的復(fù)制,這是它們致病機(jī)制中的一個特征。在之前的研究中,我們發(fā)現(xiàn)人類腸道病毒71型(EV-A71)和柯薩奇病毒A16(CA16)都可以引起宿主細(xì)胞S期阻滯以促進(jìn)其本身的病毒復(fù)制。然而EV-D68是否存在潛在的細(xì)胞周期操控能力,且其對細(xì)胞周期的操控能力是否與其最近爆發(fā)時的強(qiáng)致病性及致死性有關(guān)等問題到目前為止都沒有被研究。目的:在本次研究中,我們針對不同細(xì)胞周期狀態(tài)對EV-D68復(fù)制的影響及EV-D68對宿主細(xì)胞周期的操控能力進(jìn)行了探討。以求進(jìn)一步增加對EV-D68致病機(jī)理的理解,并且盡力為日后EV-D68感染相關(guān)疾病的預(yù)防及特異性抗病毒治療提供潛在的靶點(diǎn)。方法:為了研究不同細(xì)胞周期狀態(tài)對EV-D68復(fù)制的影響及EV-D68病毒對宿主細(xì)胞的細(xì)胞周期方面的影響,我們以無血清的DMEM培養(yǎng)RD細(xì)胞使細(xì)胞處于G0/G1期同步化狀態(tài),以0.85 m M的胸腺嘧啶核苷處理RD細(xì)胞使其處于S期同步化狀態(tài),且以25 ng/ml的諾考達(dá)唑處理細(xì)胞使其處于G2/M期同步化狀態(tài)。在實(shí)驗過程中,我們通過流式細(xì)胞術(shù)來測定宿主細(xì)胞的細(xì)胞周期曲線,且通過Mod Fit LT軟件來分析處于不同細(xì)胞周期階段的細(xì)胞占總細(xì)胞數(shù)的百分比。在基因水平,我們通過實(shí)時定量PCR技術(shù)測定RD細(xì)胞內(nèi)EV-D68的RNA水平及各種細(xì)胞周期相關(guān)蛋白的m RNA表達(dá)水平。為了測定病毒的毒力,我們以滴定法測定上清液中的子代病毒。我們通過半定量的蛋白免疫印跡方法(western blot)對EV-D68感染后細(xì)胞周期相關(guān)蛋白的調(diào)節(jié)進(jìn)行了分析,且利用Elisa試劑盒對各種細(xì)胞周期相關(guān)蛋白的表達(dá)量進(jìn)行了測定以驗證蛋白免疫印跡所得結(jié)果的正確性。結(jié)果:實(shí)驗結(jié)果表明,同步化宿主細(xì)胞周期到G0/G1期可以促進(jìn)EV-D68擴(kuò)增,S期同步化沒有促進(jìn)病毒擴(kuò)增的能力,但G2/M期同步化可以抑制病毒的復(fù)制。無論是最早被分離的EV-D68的Fermon株,還是目前正在全球范圍內(nèi)流行的EV-D68的幾株流行株都可以調(diào)控宿主細(xì)胞的細(xì)胞周期使其發(fā)生G0/G1期阻滯,進(jìn)而為EV-D68病毒的復(fù)制提供最適宜的環(huán)境。從蛋白質(zhì)表達(dá)水平和/或相應(yīng)基因的m RNA的表達(dá)水平上可以發(fā)現(xiàn),EV-D68對細(xì)胞周期的調(diào)節(jié)與細(xì)胞周期蛋白及細(xì)胞周期蛋白依賴性激酶表達(dá)的相關(guān)作用有關(guān)。此外,我們還發(fā)現(xiàn),EV-D68的病毒非結(jié)構(gòu)蛋白3D可以阻止細(xì)胞周期由G0/G1期進(jìn)入S期。有趣的是,EV-D68與EV-A71雖同為腸道病毒,但G0/G1期同步化并不能促進(jìn)EV-A71的復(fù)制,相反G0/G1期同步化卻抑制EV-A71的復(fù)制。然而,這兩種病毒卻存在一個相似點(diǎn):G2/M期同步化對這兩種病毒的復(fù)制及活性均起到抑制作用,這為腸道病毒的常規(guī)治療提供了一個靶點(diǎn)和方向。這些結(jié)果進(jìn)一步的解釋了腸道病毒特別是EV-D68的致病機(jī)制,且為日后EV-D68相關(guān)疾病的預(yù)防和治療提供了潛在的策略。結(jié)論:不同于對EV-A71的研究,本次研究的結(jié)果表明EV-D68顯示出了增加宿主細(xì)胞中G0/G1期細(xì)胞所占百分比的顯著能力,且G0/G1期是EV-D68進(jìn)行復(fù)制的最適宜的環(huán)境。且我們還發(fā)現(xiàn)G2/M期阻滯既可以抑制EV-D68的復(fù)制也可以抑制EV-A71的復(fù)制。因此,我們推測誘導(dǎo)G2/M阻滯的藥物可以被認(rèn)為是抑制不同類型的腸道病毒感染的通用抗病毒療法。這為將來抗腸道病毒及抗EV-D68藥物的發(fā)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.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R373.2

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