微管骨架在登革病毒感染中作用的初步研究

发布时间：2018-03-22 04:40

本文选题：登革病毒　切入点：ECV304　出处：《第三军医大学》2005年硕士论文　论文类型：学位论文

【摘要】：登革病毒(dengue viruses, DV)是黄病毒属(Flavivirus)的单股正链RNA 病毒,广泛流行于热带和亚热带地区,主要通过埃及伊蚊和白纹伊蚊传播,引起人类登革热(classical dengue fever, DF)和登革出血热/登革休克综合征(dengue haemorrhagic fever /dengue shock syndrome, DHF/DSS)。近年来,在世界范围内和我国南方及东南亚,DHF/DSS 的发病率有明显增加之趋势,可见登革病毒感染已是严重的公共卫生问题。登革病毒完整的感染周期分3 个步骤:进入宿主细胞、生物合成及组装释放。近年来的大量研究认为微管骨架在病毒和病毒蛋白从细胞周边向细胞核方向的逆行性运输以及从细胞中心向细胞周边区域的顺行性运输的转运过程中起着重要的作用,因而推测,登革病毒可能也利用现成的微管骨架,以完成其在细胞内的移行。微管研究中最常用的药物有2 类:一类阻止微管蛋白的添加,从而阻断微管的组装,并引起原有微管的解聚,如秋水酰胺,长春花碱,诺考哒唑等;另一类为稳定微管的药物,如紫杉醇,紫杉酚。本研究以脐静脉来源的ECV304 细胞和人肝癌细胞株HepG2 细胞为研究对象,探讨了登革病毒在与宿主细胞相互作用过程中微管的变化,同时选用了秋水酰胺(demecocline),诺考哒唑(nocodazole) 和紫杉醇(paclitaxel)等药物,在病毒复制的不同环节干扰感染实验,期望从不同的角度分析微管骨架在登革病毒复制过程中的作用。本研究的主要结果和结论如下: 1. 登革病毒在ECV304 细胞中的增殖规律以MOI=1 感染ECV304 细胞,感染后72 h 达高峰,滴度约为106PFU/ml,随之出现渐降趋势。为明确病毒释放的时段,我们对感染后12 h 内培养上清的病毒滴度,进行了分时段检测,结果表明感染后4-5 h 培养细胞上清即可检测到病毒,在0、2、5、8 h,病毒滴度分别为1.1×10~3PFU/ml、1.2×10~3 PFU/ml、4.25×10~3 PFU/ml、1.05×10~4 PFU/ml,因而我们认为感染后5-8 h 可能是登革病毒从ECV304 细胞释放的关键时段。 2.登革病毒感染引起微管骨架排列变化,登革病毒抗原与微管共染正常情况下,微管由核向外呈辐射状分布,核周的微管组织中心(microtubule organizing center, MTOC)呈星形,位于胞核一侧, 荧光强度较强。本实验发现感染后24 h,微管平均荧光强度减弱,排列紊乱,主要表现为(1)辐射状分布的微管变成无
[Abstract]:Dengue virus (DVV), a single-stranded positive RNA virus of the genus Flavivirus, is widely spread in tropical and subtropical regions and is mainly transmitted through Aedes aegypti and Aedes albopictus. The incidence of dengue haemorrhagic fever / dengue shock syndromes (DHFR / DSS) and dengue hemorrhagic fever / dengue shock syndrome (DHF / DDS / DHF / DSS) in humans has increased significantly in recent years. It can be seen that dengue virus infection is a serious public health problem. The complete infection cycle of dengue virus consists of three steps: entering the host cell, Biosynthesis and assembly release. Recent studies have shown that microtubule cytoskeleton transports retrograde transport of virus and viral proteins from the periphery of the cell to the nucleus and from the center of the cell to the peripheral region of the cell. It plays an important role in the process of transportation. Therefore, it is speculated that dengue virus may also use a ready-made microtubule skeleton to complete its intracellular migration. There are two kinds of drugs most commonly used in microtubule studies: one is to block the addition of tubulin, thus blocking the assembly of microtubules. And caused the depolymerization of the original microtubules, such as autumn water amide, vincristine, noconazole, etc. The other kind of microtubule stable drugs, such as paclitaxel, In this study, ECV304 cells from umbilical vein and HepG2 cells from human hepatoma cells were used to study the changes of microtubules during the interaction between dengue virus and host cells. At the same time, some drugs, such as Akihuanamide demecocline, nocodazolein and paclitaxel, were used to interfere with the infection in different links of virus replication, and to analyze the role of microtubule skeleton in the replication of dengue virus from different angles. The main findings and conclusions of this study are as follows:. 1. Proliferation of dengue virus in ECV304 cells. The ECV304 cells infected with MOI=1 reached the peak at 72 h after infection, and the titer was about 106 PFU / ml. In order to determine the time of virus release, we detected the titer of the supernatant within 12 hours after infection. The results showed that the virus could be detected in the supernatant of cultured cells 4-5 hours after infection. The titer of the virus was 1.1 脳 10 ~ (-3) PFU / ml = 1.2 脳 10 ~ (-3) PFU / ml = 1.25 脳 10 ~ (-3) PFU / ml ~ (1.05) 脳 10 ~ (4) PFU / ml, respectively. Therefore, we think that 5-8 h after infection may be the key time for dengue virus to be released from ECV304 cells. 2. Changes in the arrangement of microtubule cytoskeleton caused by dengue virus infection and co-staining of dengue virus antigen with microtubule. Under normal conditions, the microtubule organizing center (MTOC) of the surrounding microtubules is starlike, and the fluorescence intensity is strong at one side of the nucleus. It was found that the average fluorescence intensity of the microtubules decreased at 24 h after infection, and the arrangement of the microtubules was disordered. The microtubules with radiative distribution become non-existent.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R373

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