本文選題：活細(xì)胞　切入點(diǎn)：脊髓灰質(zhì)炎病毒正鏈RNA　出處：《中國(guó)科學(xué)院研究生院（武漢病毒研究所）》2005年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：病毒核酸在寄主細(xì)胞內(nèi)定位和運(yùn)動(dòng)的監(jiān)測(cè)分析對(duì)于理解病毒與宿主細(xì)胞間的相互作用具有重要意義。但是迄今為止,要實(shí)現(xiàn)病毒核酸在寄主細(xì)胞內(nèi)定位和運(yùn)動(dòng)的分析,特別是要實(shí)現(xiàn)病毒核酸在活細(xì)胞內(nèi)的動(dòng)態(tài)學(xué)及其控制機(jī)制的分析,還存在方法學(xué)上的不足和挑戰(zhàn)。 利用分子信標(biāo)技術(shù)和脊髓灰質(zhì)炎病毒-Vero 細(xì)胞作為模型病毒-細(xì)胞系統(tǒng),本研究建立了一種在活的寄主細(xì)胞內(nèi)實(shí)現(xiàn)病毒核酸可視化的方法。當(dāng)把目標(biāo)RNA特異性的分子信標(biāo)探針引入脊髓灰質(zhì)炎病毒感染的Vero 細(xì)胞內(nèi)時(shí),內(nèi)源的脊髓灰質(zhì)炎病毒的正鏈RNA 可以在活細(xì)胞內(nèi)被標(biāo)記照亮。在熒光顯微鏡下可以直接監(jiān)測(cè)脊髓灰質(zhì)炎病毒的正鏈RNA 在活細(xì)胞內(nèi)的定位和運(yùn)動(dòng)。這一結(jié)果也證明了分子信標(biāo)是實(shí)現(xiàn)病毒核酸在活細(xì)胞內(nèi)可視化的有力工具。 運(yùn)用長(zhǎng)時(shí)間活細(xì)胞熒光影像捕獲和熒光漂白恢復(fù)等技術(shù)手段,研究了脊髓灰質(zhì)炎病毒正鏈RNA 在活細(xì)胞內(nèi)的行為特征。在病毒感染后的不同時(shí)間,脊髓灰質(zhì)炎病毒正鏈RNA 在寄主活細(xì)胞內(nèi)展示不同的分布模式。實(shí)時(shí)監(jiān)測(cè)發(fā)現(xiàn)脊髓灰質(zhì)炎病毒正鏈RNA 在Vero 細(xì)胞內(nèi)的分布變化是一個(gè)特征性的RNA 移位過(guò)程。由于這一移位過(guò)程類(lèi)似于脊髓灰質(zhì)炎病毒誘導(dǎo)的特異性膜泡在寄主細(xì)胞內(nèi)的重排過(guò)程,我們把這種RNA 移位也稱(chēng)為RNA 的重排。脊髓灰質(zhì)炎病毒正鏈RNA 特征性的移位和重排需要寄主細(xì)胞完整的微管網(wǎng)絡(luò),破壞微管網(wǎng)絡(luò)可以改變RNA 的分布模式。熒光漂白恢復(fù)檢測(cè)顯示約有49.4 ±3.2%的脊髓灰質(zhì)炎病毒正鏈RNA 在其分布區(qū)域內(nèi)部可以自由擴(kuò)散運(yùn)動(dòng)(擴(kuò)散系數(shù)為9.6 ±1.6×10~(-10) cm~2/s),其余的50.5 ±2.9%在FRAP 測(cè)定期間幾乎靜止不動(dòng),而只是隨著整個(gè)RNA 分布區(qū)域的變化緩慢移動(dòng)�；罴�(xì)胞內(nèi)的研究揭示了脊髓灰質(zhì)炎病毒正鏈RNA 以一種受限制和自由擴(kuò)散相混合的復(fù)雜的分布和運(yùn)動(dòng)機(jī)制存在于寄主細(xì)胞。這種機(jī)制主要與寄主細(xì)胞的微管和病毒誘導(dǎo)的膜結(jié)構(gòu)有關(guān)。這一病毒核酸在活的寄主細(xì)胞的動(dòng)態(tài)行為的研究也讓我們對(duì)脊髓灰質(zhì)炎病毒和寄主細(xì)胞的相互作用有了更深的理解。 通過(guò)電子顯微鏡的觀察,發(fā)現(xiàn)脊髓灰質(zhì)炎病毒誘導(dǎo)的膜重排過(guò)程也與微管組織有關(guān)。結(jié)合文獻(xiàn)資料和本研究的發(fā)現(xiàn),我們猜想脊髓灰質(zhì)炎感染細(xì)胞后,伴隨膜重排的過(guò)程,寄主細(xì)胞內(nèi)還可能有一個(gè)特征性的微管重排過(guò)程。
[Abstract]:The localization and monitoring of viral nucleic acids in host cells are important for understanding the interaction between virus and host cells, but so far, the localization and movement of viral nucleic acids in host cells have been analyzed. Especially in order to realize the dynamic analysis of viral nucleic acid in living cells and its control mechanism, there are still shortcomings and challenges in methodology. Using molecular beacons and poliovirus-Vero cells as model virus-cell systems, This study established a method to visualize viral nucleic acids in live host cells. When the target RNA specific molecular beacon probe was introduced into poliovirus infected Vero cells, The positive strand RNA of endogenous poliovirus can be illuminated in living cells. The localization and movement of positive strand RNA of poliovirus in living cells can be directly monitored under fluorescence microscope. Molecular beacons are powerful tools for visualizing viral nucleic acids in living cells. The behavioral characteristics of poliovirus positive strand RNA in living cells were studied by means of long time living cell fluorescence capture and fluorescence bleaching recovery. Poliovirus positive strand RNA showed different distribution patterns in host living cells. Real-time surveillance showed that the distribution of poliovirus positive strand RNA in Vero cells was a characteristic RNA translocation process. The translocation process is similar to the rearrangement of specific membrane vesicles in host cells induced by poliovirus. We call this RNA translocation also known as rearrangement of RNA. The characteristic shift and rearrangement of positive strand RNA of poliovirus require a complete microtubule network of host cells. Destroying the microtubule network can change the distribution pattern of RNA. Fluorescence bleaching recovery assay shows that about 49.4 鹵3.2% of poliovirus positive chain RNA can move freely within its distribution region (diffusion coefficient is 9.6 鹵1.6 脳 10 ~ (-10)) cm ~ (2) / s ~ (-1), and the rest of the. 50.5 鹵2.9% was almost stationary during the determination of FRAP, Studies in living cells revealed that poliovirus positive strand RNA existed in the host as a complex distribution and motor mechanism of limited and free diffusion. This mechanism is mainly related to the microtubules of the host cell and the membrane structure induced by the virus. The study of the dynamic behavior of the viral nucleic acid in the living host cell also allows us to interact with the host cell and the poliovirus. The role has been further understood. By electron microscope observation, we found that the membrane rearrangement induced by poliovirus is also related to microtubule tissue. In combination with the literature and findings of this study, we suspect that the process of membrane rearrangement after poliomyelitis infection is accompanied by membrane rearrangement. There may also be a characteristic microtubule rearrangement in host cells.
【學(xué)位授予單位】：中國(guó)科學(xué)院研究生院（武漢病毒研究所）
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2005
【分類(lèi)號(hào)】：R373

【共引文獻(xiàn)】

相關(guān)期刊論文 前2條

1 安瑞芳;薛艷;張永愛(ài);賀大林;趙軍;王新陽(yáng);楊麗麗;;宮頸癌細(xì)胞株survivin mRNA表達(dá)的分子信標(biāo)檢測(cè)[J];腫瘤預(yù)防與治療;2008年04期

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1 李軍;分子信標(biāo)熒光探針用于遺傳物質(zhì)的超靈敏檢測(cè)及超微型DNA、pH傳感器的研究[D];湖南大學(xué);2002年

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1 陳歡;用于生物熒光標(biāo)記的稀土上轉(zhuǎn)換發(fā)光納米顆粒的制備與性質(zhì)研究[D];吉林大學(xué);2012年

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