本文選題：登革病毒 + ECV304細胞��； 參考：《第三軍醫(yī)大學》2011年碩士論文

【摘要】：登革病毒(dengue virus,DV)屬于黃病毒屬、是一種包膜的單股正鏈RNA病毒。根據包膜蛋白的抗原性不同,可將登革病毒分為四個血清型,即DV1~4。DV以蚊蟲為主要傳播媒介廣泛流行于熱帶和亞熱帶地區(qū)。每年,登革病毒會導致數百萬人感染,引起登革熱(dengue fever,DF)和登革出血熱/登革休克綜合癥(dengue hemorrhagic fever/ dengueshock syndrome, DHF/DSS)。DF是自限性發(fā)熱性疾病。而DHF/DSS則是威脅患者生命的重癥,其主要特征是血管通透性顯著增加,導致血漿滲漏。每年大約有50萬DHF/DSS患者,如未及時治療,病死率可上升至50%。因而對其致病機理和預防手段的研究已成為亟待解決的前沿課題。 研究表明,宿主細胞骨架在病毒的感染過程中發(fā)揮重要作用,而構成細胞骨架的主要成分微絲、微管、中間纖維在不同病毒的感染過程中分別扮演著不同的角色。我們小組前期實驗證實微絲和波形蛋白纖維在DV的感染及復制過程中發(fā)揮著重要作用。DV的感染可以引起微絲骨架的改變,用微絲藥物破壞其聚合和解離的平衡,可以導致DV感染受到抑制說明微絲聚合和解離的平衡對DV的感染具有重要意義。而調節(jié)微絲骨架的關鍵因子是Rho GTP酶,作為分子開關Rho GTP酶在無活性的GDP和有活性的GTP兩種形式間循環(huán),調節(jié)細胞的形態(tài)、生長、運動以及細胞周期等。Rho GTP酶家族中研究的比較多的有3個成員:RhoA、Cdc42和Rac1。我們對波形蛋白纖維的研究發(fā)現(xiàn),ECV304細胞在感染DV2后,細胞中的波形蛋白會發(fā)生重排,重排的波形蛋白從細胞邊緣回縮、環(huán)繞于細胞核周圍,并與病毒抗原共存,用丙烯酰胺長時間作用細胞后,破壞波形蛋白會影響DV2的復制增殖。目前研究發(fā)現(xiàn)波形蛋白纖維的重排是由激酶磷酸化所致,而RhoA及其下游激酶ROCK (Rho associated coiled-coil forming protein kinase)在波形蛋白纖維磷酸化中扮演著重要角色。為此,本課題以RhoA/ ROCK通路為研究對象,采用構建RhoA突變體及RhoA和ROCK特異性抑制劑干擾RhoA/ ROCK通路的方法,觀察對DV感染與復制的不同環(huán)節(jié)的影響,從而為解析DV感染的分子機制提供理論依據,為預防和控制DV感染提供新思路。本研究的主要實驗內容和結果如下: 1.利用突變體觀察RhoA功能的異常對DV病毒感染的影響 本實驗用DV2感染ECV304細胞及穩(wěn)定表達RhoA突變體的ECV304細胞株:ECV304N、ECVWtRhoA、ECVV14RhoA和ECVN19RhoA(MOI=1),通過病毒噬斑計數法分別檢測感染后1 h細胞內和24h細胞內外的病毒滴度。結果顯示在病毒感染1小時后ECVN19RhoA、ECVWtRhoA、ECVV14RhoA細胞內的病毒滴度均低于ECV304和ECV304N,其中ECVV14RhoA、ECVWtRhoA下降比較明顯,而ECVN19RhoA下降較少,相較于ECV304N的病毒滴度分別下降了49.71%、51.76%、20.59%。在病毒感染24小時后,細胞上清中的病毒滴度ECVV14RhoA、ECVWtRhoA和ECVN19RhoA均低于ECV304N和ECV304,其中ECVV14RhoA、ECVWtRhoA下降比較明顯,而ECVN19RhoA下降不明顯,相較于ECV304N的病毒滴度分別下降了76.6%、77.2%、23%;細胞內病毒滴度的變化與上清趨勢相符,即ECVV14RhoA、ECVWtRhoA下降比較明顯,而ECVN19RhoA下降不明顯。相較于ECV304N的病毒滴度分別下降了58.75%、62.32%、20.29%。結果提示:RhoA功能的異常對DV2的感染有明顯的抑制作用。 2.利用C3轉移酶抑制RhoA活性觀察對DV感染的影響 利用C3轉移酶抑制ECV304細胞內RhoA活化后實施感染實驗,收集病毒穿入細胞1 h時的細胞樣本和感染后24 h培養(yǎng)上清及細胞樣本進行病毒滴度檢測,結果顯示:藥物組病毒穿入1h后細胞內的病毒滴度相較于對照組的病毒滴度下降了88.46%;藥物組病毒感染24h培養(yǎng)上清和細胞內的病毒滴度相較于對照組分別下降了12.52%、82.35%。與免疫熒光及共聚焦顯微鏡觀察到的結果相符,即藥物處理組病毒抗原陽性的細胞數目明顯低于對照組。結果說明抑制RhoA活化可明顯抑制DV穿入宿主細胞及DV的復制增殖。 3.采用G-lisa檢測DV病毒感染過程中RhoA分子活性的變化 用滅活DV2(56℃,30min)和DV2分別感染ECV304細胞。在感染30min,1h,8h及24時收集細胞樣品,采用G-LISATM RhoA Activation Assay Biochem KitTM(Cytoskeleton)試劑盒檢測胞內RhoA分子的活性。結果顯示:在感染30min及1h時RhoA活性較對照組有顯著升高,其中感染30min時最為明顯,感染后1h開始下降。而在感染8h和24h后RhoA分子活性較對照組沒有明顯變化。說明RhoA活性主要在病毒穿入細胞的過程中被激活,而在病毒的復制增殖過程中沒有變化。 4.利用Y-27632抑制ROCK活性觀察對DV感染的影響 使用ROCK活性抑制劑Y-27632抑制ROCK活性后實施感染實驗,收集感染后8 h和24 h培養(yǎng)上清和細胞樣本進行病毒滴度檢測,結果顯示:與對照組相比藥物處理組(6個濃度處理)細胞上清和細胞樣本中病毒滴度均沒有明顯變化。與免疫熒光及共聚焦顯微鏡觀察到結果一致,即藥物處理組與對照組之間病毒抗原陽性的細胞數目沒有明顯差異。說明抑制ROCK活性對DV的復制增殖無明顯影響。 綜上所述,RhoA在DV感染過程中具有重要作用,DV在進入ECV304細胞的過程中激活RhoA。上述實驗結果為深入理解DV與宿主細胞的相互作用、闡明DV的致病機制提供了重要的實驗依據。
[Abstract]:Dengue virus (DV) belongs to the genus yellows and is a coated single strand RNA virus. According to the antigenicity of the enveloped protein, dengue virus can be divided into four serotypes. That is, DV1~4.DV is widely prevalent in the tropical and subtropical areas with the main vectors of mosquitoes. Dengue fever (dengue fever, DF) and dengue hemorrhagic fever / dengue shock syndrome (dengue hemorrhagic fever/ dengueshock syndrome, DHF/DSS).DF are self limiting febrile diseases. DHF/DSS is a serious threat to patients' life, whose main feature is the significant increase in vascular permeability, leading to plasma leakage. About 500 thousand DHF/DSS patients each year, Without timely treatment, the mortality rate can rise to 50%.. Therefore, the research on its pathogenesis and preventive measures has become an urgent topic to be solved.
Studies have shown that the host cytoskeleton plays an important role in the infection process of the virus, and the main components of the cytoskeleton, microfilament, microtubule, and intermediate fibers play different roles in the infection process of different viruses. Our group earlier experiments confirmed that microfilament and fibrin fiber play a role in the infection and replication of DV. The infection of.DV can cause the change of the microfilament skeleton, and the balance between the polymerization and dissociation of the microfilament drugs can lead to the inhibition of DV infection, which indicates that the balance of microfilament polymerization and dissociation is of great significance to the infection of DV. The key factor for regulating the microfilament skeleton is the Rho GTP enzyme, as the molecular switch Rho GTP enzyme is not alive. There are more than 3 members of the.Rho GTP enzyme family in the.Rho GTP enzyme family that regulates the form, growth, movement, and cell cycle of the two forms of the.Rho GTP enzyme in the cell morphology, growth, movement, and cell cycle. The study of vimentin fibers in RhoA, Cdc42 and Rac1. found that the vimentin in the cells would rearrange and rearrange after the infection of DV2. Vimentin is retracted from the edge of the cell and surrounds the nucleus and coexists with the virus antigen. After a long time action of the cells with acrylamide, the destruction of vimentin will affect the replication and proliferation of DV2. At present, the rearrangement of vimentin fibers is caused by kinase phosphorylation, while RhoA and its downstream kinase ROCK (Rho associated coiled-coi) L forming protein kinase (protein kinase) plays an important role in the phosphorylation of vimentin fiber. Therefore, this subject takes the RhoA/ ROCK pathway as the research object, and uses the method of constructing RhoA mutants and RhoA and ROCK specific inhibitors to interfere with RhoA/ ROCK pathway to observe the influence of the different links on the infection and replication of DV, so as to analyze the infection. The molecular mechanism provides a theoretical basis for the prevention and control of DV infection. The main contents and results of this study are as follows:
1. observe the effect of abnormal RhoA function on DV virus infection by mutants.
In this experiment, DV2 infected ECV304 cells and ECV304 cells that stably expressed RhoA mutants: ECV304N, ECVWtRhoA, ECVV14RhoA and ECVN19RhoA (MOI=1). Virus titers were detected by virus plaque counting method in 1 h cells and 24h cells after infection. The titer of the virus was lower than that of ECV304 and ECV304N, of which ECVV14RhoA, ECVWtRhoA decreased significantly, while ECVN19RhoA decreased less and the virus titer of ECV304N decreased by 49.71%, 51.76% respectively. The virus titer in the cell supernatant was ECVV14RhoA, ECVWtRhoA and ECVN19RhoA were lower than ECV304N and ECV304, after 24 hours of virus infection. The decrease of ECVV14RhoA and ECVWtRhoA was obvious, but the decrease of ECVN19RhoA was not obvious, and the virus titer of ECV304N decreased by 76.6%, 77.2%, 23%, and the change of virus titer in cell line was consistent with the trend of the supernatant, that is, ECVV14RhoA, ECVWtRhoA drop is more obvious, but the drop of ECVN19RhoA is not obvious. The virus titer of ECV304N is lower than that of ECV304N, respectively. A decrease of 58.75%, 62.32%, and 20.29%. results suggest that the abnormality of RhoA function has an obvious inhibitory effect on DV2 infection.
2. the effect of C3 transferase inhibition on RhoA activity on DV infection.
Using C3 transferase to inhibit RhoA activation in ECV304 cells, the infection experiment was carried out. The cell samples and the 24 h culture supernatant and the cell samples were collected to detect the virus titer. The results showed that the virus titer in the drug group was 88.46% lower than that of the control group after the virus penetrated 1H. The virus titer in the 24h culture supernatant and in the cell was decreased by 12.52% compared with the control group. 82.35%. was in accordance with the results observed by immunofluorescence and confocal microscopy, that is, the number of positive cells in the drug treatment group was significantly lower than that of the control group. The results indicated that the inhibition of RhoA activation could significantly inhibit the penetration of DV into the host. Replication and proliferation of cells and DV.
3. detect the changes of RhoA activity during DV virus infection by G-lisa.
ECV304 cells were infected with inactivated DV2 (56, 30min) and DV2 respectively. The cell samples were collected at 30min, 1H, 8h and 24. The activity of G-LISATM RhoA Activation Assay Biochem assay kit was used to detect the activity of intracellular molecules. The results showed that the activity of the cells was significantly higher than that of the control group. It was most obvious that 1H began to decline after infection, but there was no significant change in the activity of RhoA molecules after infection with 8h and 24h, indicating that the activity of RhoA was activated mainly during the process of virus penetrating the cells, but not in the process of replication and proliferation of the virus.
4. inhibition of ROCK activity by Y-27632 on DV infection.
The infection experiment was carried out after the inhibition of the activity of the ROCK active inhibitor Y-27632 on ROCK activity. The virus titer of the 8 h and 24 h culture supernatants and cell samples after infection was collected. The results showed that there was no significant change in the titer of the cell supernatant and cell samples in the drug treatment group (6 concentration treatment) compared with the control group. The results of the focal microscope were consistent, that is, there was no significant difference in the number of positive cells between the drug treatment group and the control group. It showed that the inhibition of ROCK activity had no significant effect on the replication and proliferation of DV.
In summary, RhoA plays an important role in the process of DV infection. The results of DV activation of RhoA. in the process of entering ECV304 cells provide an important experimental basis for understanding the interaction between DV and host cells, and clarifying the pathogenesis of DV.

【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】：2011
【分類號】：R373

【共引文獻】

相關期刊論文 前2條

1 陳煒;高娜;王嘉麗;張俊磊;田衍平;安靜;;穩(wěn)定過表達p50基因細胞株的構建及鑒定[J];局解手術學雜志;2009年02期

2 宋富強;陳煒;王嘉麗;張俊磊;胡曉梅;;RhoA突變體的構建及穩(wěn)定表達[J];免疫學雜志;2010年10期

，

本文編號：1784676