活细胞内脊髓灰质炎病毒正链RNA可视化研究

发布时间：2018-03-12 11:02

本文选题：活细胞　切入点：脊髓灰质炎病毒正链RNA　出处：《中国科学院研究生院（武汉病毒研究所）》2005年博士论文　论文类型：学位论文

【摘要】：病毒核酸在寄主细胞内定位和运动的监测分析对于理解病毒与宿主细胞间的相互作用具有重要意义。但是迄今为止,要实现病毒核酸在寄主细胞内定位和运动的分析,特别是要实现病毒核酸在活细胞内的动态学及其控制机制的分析,还存在方法学上的不足和挑战。利用分子信标技术和脊髓灰质炎病毒-Vero 细胞作为模型病毒-细胞系统,本研究建立了一种在活的寄主细胞内实现病毒核酸可视化的方法。当把目标RNA特异性的分子信标探针引入脊髓灰质炎病毒感染的Vero 细胞内时,内源的脊髓灰质炎病毒的正链RNA 可以在活细胞内被标记照亮。在荧光显微镜下可以直接监测脊髓灰质炎病毒的正链RNA 在活细胞内的定位和运动。这一结果也证明了分子信标是实现病毒核酸在活细胞内可视化的有力工具。运用长时间活细胞荧光影像捕获和荧光漂白恢复等技术手段,研究了脊髓灰质炎病毒正链RNA 在活细胞内的行为特征。在病毒感染后的不同时间,脊髓灰质炎病毒正链RNA 在寄主活细胞内展示不同的分布模式。实时监测发现脊髓灰质炎病毒正链RNA 在Vero 细胞内的分布变化是一个特征性的RNA 移位过程。由于这一移位过程类似于脊髓灰质炎病毒诱导的特异性膜泡在寄主细胞内的重排过程,我们把这种RNA 移位也称为RNA 的重排。脊髓灰质炎病毒正链RNA 特征性的移位和重排需要寄主细胞完整的微管网络,破坏微管网络可以改变RNA 的分布模式。荧光漂白恢复检测显示约有49.4 ±3.2%的脊髓灰质炎病毒正链RNA 在其分布区域内部可以自由扩散运动(扩散系数为9.6 ±1.6×10~(-10) cm~2/s),其余的50.5 ±2.9%在FRAP 测定期间几乎静止不动,而只是随着整个RNA 分布区域的变化缓慢移动。活细胞内的研究揭示了脊髓灰质炎病毒正链RNA 以一种受限制和自由扩散相混合的复杂的分布和运动机制存在于寄主细胞。这种机制主要与寄主细胞的微管和病毒诱导的膜结构有关。这一病毒核酸在活的寄主细胞的动态行为的研究也让我们对脊髓灰质炎病毒和寄主细胞的相互作用有了更深的理解。通过电子显微镜的观察,发现脊髓灰质炎病毒诱导的膜重排过程也与微管组织有关。结合文献资料和本研究的发现,我们猜想脊髓灰质炎感染细胞后,伴随膜重排的过程,寄主细胞内还可能有一个特征性的微管重排过程。
[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 卤3.2% of poliovirus positive chain RNA can move freely within its distribution region (diffusion coefficient is 9.6 卤1.6 脳 10 ~ (-10)) cm ~ (2) / s ~ (-1), and the rest of the. 50.5 卤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.
【学位授予单位】：中国科学院研究生院（武汉病毒研究所）
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R373

【共引文献】