RhoA分子在登革2型病毒感染过程中的作用研究

发布时间：2018-04-22 00:01

本文选题：登革病毒 + ECV304细胞　；参考：《第三军医大学》2011年硕士论文

【摘要】：登革病毒(dengue virus,DV)属于黄病毒属、是一种包膜的单股正链RNA病毒。根据包膜蛋白的抗原性不同,可将登革病毒分为四个血清型,即DV1~4。DV以蚊虫为主要传播媒介广泛流行于热带和亚热带地区。每年,登革病毒会导致数百万人感染,引起登革热(dengue fever,DF)和登革出血热/登革休克综合症(dengue hemorrhagic fever/ dengueshock syndrome, DHF/DSS)。DF是自限性发热性疾病。而DHF/DSS则是威胁患者生命的重症,其主要特征是血管通透性显著增加,导致血浆渗漏。每年大约有50万DHF/DSS患者,如未及时治疗,病死率可上升至50%。因而对其致病机理和预防手段的研究已成为亟待解决的前沿课题。研究表明,宿主细胞骨架在病毒的感染过程中发挥重要作用,而构成细胞骨架的主要成分微丝、微管、中间纤维在不同病毒的感染过程中分别扮演着不同的角色。我们小组前期实验证实微丝和波形蛋白纤维在DV的感染及复制过程中发挥着重要作用。DV的感染可以引起微丝骨架的改变,用微丝药物破坏其聚合和解离的平衡,可以导致DV感染受到抑制说明微丝聚合和解离的平衡对DV的感染具有重要意义。而调节微丝骨架的关键因子是Rho GTP酶,作为分子开关Rho GTP酶在无活性的GDP和有活性的GTP两种形式间循环,调节细胞的形态、生长、运动以及细胞周期等。Rho GTP酶家族中研究的比较多的有3个成员:RhoA、Cdc42和Rac1。我们对波形蛋白纤维的研究发现,ECV304细胞在感染DV2后,细胞中的波形蛋白会发生重排,重排的波形蛋白从细胞边缘回缩、环绕于细胞核周围,并与病毒抗原共存,用丙烯酰胺长时间作用细胞后,破坏波形蛋白会影响DV2的复制增殖。目前研究发现波形蛋白纤维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)在波形蛋白纤维磷酸化中扮演着重要角色。为此,本课题以RhoA/ ROCK通路为研究对象,采用构建RhoA突变体及RhoA和ROCK特异性抑制剂干扰RhoA/ ROCK通路的方法,观察对DV感染与复制的不同环节的影响,从而为解析DV感染的分子机制提供理论依据,为预防和控制DV感染提供新思路。本研究的主要实验内容和结果如下: 1.利用突变体观察RhoA功能的异常对DV病毒感染的影响本实验用DV2感染ECV304细胞及稳定表达RhoA突变体的ECV304细胞株:ECV304N、ECVWtRhoA、ECVV14RhoA和ECVN19RhoA(MOI=1),通过病毒噬斑计数法分别检测感染后1 h细胞内和24h细胞内外的病毒滴度。结果显示在病毒感染1小时后ECVN19RhoA、ECVWtRhoA、ECVV14RhoA细胞内的病毒滴度均低于ECV304和ECV304N,其中ECVV14RhoA、ECVWtRhoA下降比较明显,而ECVN19RhoA下降较少,相较于ECV304N的病毒滴度分别下降了49.71%、51.76%、20.59%。在病毒感染24小时后,细胞上清中的病毒滴度ECVV14RhoA、ECVWtRhoA和ECVN19RhoA均低于ECV304N和ECV304,其中ECVV14RhoA、ECVWtRhoA下降比较明显,而ECVN19RhoA下降不明显,相较于ECV304N的病毒滴度分别下降了76.6%、77.2%、23%;细胞内病毒滴度的变化与上清趋势相符,即ECVV14RhoA、ECVWtRhoA下降比较明显,而ECVN19RhoA下降不明显。相较于ECV304N的病毒滴度分别下降了58.75%、62.32%、20.29%。结果提示:RhoA功能的异常对DV2的感染有明显的抑制作用。 2.利用C3转移酶抑制RhoA活性观察对DV感染的影响利用C3转移酶抑制ECV304细胞内RhoA活化后实施感染实验,收集病毒穿入细胞1 h时的细胞样本和感染后24 h培养上清及细胞样本进行病毒滴度检测,结果显示:药物组病毒穿入1h后细胞内的病毒滴度相较于对照组的病毒滴度下降了88.46%;药物组病毒感染24h培养上清和细胞内的病毒滴度相较于对照组分别下降了12.52%、82.35%。与免疫荧光及共聚焦显微镜观察到的结果相符,即药物处理组病毒抗原阳性的细胞数目明显低于对照组。结果说明抑制RhoA活化可明显抑制DV穿入宿主细胞及DV的复制增殖。 3.采用G-lisa检测DV病毒感染过程中RhoA分子活性的变化用灭活DV2(56℃,30min)和DV2分别感染ECV304细胞。在感染30min,1h,8h及24时收集细胞样品,采用G-LISATM RhoA Activation Assay Biochem KitTM(Cytoskeleton)试剂盒检测胞内RhoA分子的活性。结果显示:在感染30min及1h时RhoA活性较对照组有显著升高,其中感染30min时最为明显,感染后1h开始下降。而在感染8h和24h后RhoA分子活性较对照组没有明显变化。说明RhoA活性主要在病毒穿入细胞的过程中被激活,而在病毒的复制增殖过程中没有变化。 4.利用Y-27632抑制ROCK活性观察对DV感染的影响使用ROCK活性抑制剂Y-27632抑制ROCK活性后实施感染实验,收集感染后8 h和24 h培养上清和细胞样本进行病毒滴度检测,结果显示:与对照组相比药物处理组(6个浓度处理)细胞上清和细胞样本中病毒滴度均没有明显变化。与免疫荧光及共聚焦显微镜观察到结果一致,即药物处理组与对照组之间病毒抗原阳性的细胞数目没有明显差异。说明抑制ROCK活性对DV的复制增殖无明显影响。综上所述,RhoA在DV感染过程中具有重要作用,DV在进入ECV304细胞的过程中激活RhoA。上述实验结果为深入理解DV与宿主细胞的相互作用、阐明DV的致病机制提供了重要的实验依据。
[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.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R373

【共引文献】