本文選題：登革病毒　切入點：ECV304　出處：《第三軍醫(yī)大學》2005年碩士論文　論文類型：學位論文

【摘要】：登革病毒(dengue viruses, DV)是黃病毒屬(Flavivirus)的單股正鏈RNA 病毒,廣泛流行于熱帶和亞熱帶地區(qū),主要通過埃及伊蚊和白紋伊蚊傳播,引起人類登革熱(classical dengue fever, DF)和登革出血熱/登革休克綜合征(dengue haemorrhagic fever /dengue shock syndrome, DHF/DSS)。近年來,在世界范圍內(nèi)和我國南方及東南亞,DHF/DSS 的發(fā)病率有明顯增加之趨勢,可見登革病毒感染已是嚴重的公共衛(wèi)生問題。 登革病毒完整的感染周期分3 個步驟:進入宿主細胞、生物合成及組裝釋放。近年來的大量研究認為微管骨架在病毒和病毒蛋白從細胞周邊向細胞核方向的逆行性運輸以及從細胞中心向細胞周邊區(qū)域的順行性運輸?shù)霓D(zhuǎn)運過程中起著重要的作用,因而推測,登革病毒可能也利用現(xiàn)成的微管骨架,以完成其在細胞內(nèi)的移行。微管研究中最常用的藥物有2 類:一類阻止微管蛋白的添加,從而阻斷微管的組裝,并引起原有微管的解聚,如秋水酰胺,長春花堿,諾考噠唑等;另一類為穩(wěn)定微管的藥物,如紫杉醇,紫杉酚。本研究以臍靜脈來源的ECV304 細胞和人肝癌細胞株HepG2 細胞為研究對象,探討了登革病毒在與宿主細胞相互作用過程中微管的變化,同時選用了秋水酰胺(demecocline),諾考噠唑(nocodazole) 和紫杉醇(paclitaxel)等藥物,在病毒復制的不同環(huán)節(jié)干擾感染實驗,期望從不同的角度分析微管骨架在登革病毒復制過程中的作用。 本研究的主要結(jié)果和結(jié)論如下: 1. 登革病毒在ECV304 細胞中的增殖規(guī)律 以MOI=1 感染ECV304 細胞,感染后72 h 達高峰,滴度約為106PFU/ml,隨之出現(xiàn)漸降趨勢。為明確病毒釋放的時段,我們對感染后12 h 內(nèi)培養(yǎng)上清的病毒滴度,進行了分時段檢測,結(jié)果表明感染后4-5 h 培養(yǎng)細胞上清即可檢測到病毒,在0、2、5、8 h,病毒滴度分別為1.1×10~3PFU/ml、1.2×10~3 PFU/ml、4.25×10~3 PFU/ml、1.05×10~4 PFU/ml,因而我們認為感染后5-8 h 可能是登革病毒從ECV304 細胞釋放的關(guān)鍵時段。 2.登革病毒感染引起微管骨架排列變化,登革病毒抗原與微管共染 正常情況下,微管由核向外呈輻射狀分布,核周的微管組織中心(microtubule organizing center, MTOC)呈星形,位于胞核一側(cè), 熒光強度較強。本實驗發(fā)現(xiàn)感染后24 h,微管平均熒光強度減弱,排列紊亂,主要表現(xiàn)為(1)輻射狀分布的微管變成無
[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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R373

【共引文獻】

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本文編號：1647135