细胞ERK通路及其关键激酶MEK不同亚型在调控单纯疱疹病毒Ⅱ型复制中作用研究
发布时间：2019-03-04 19:43
【摘要】：目的： 本研究通过阐明细胞Raf/MEK/ERK(简称ERK)通路在调控单纯疱疹病毒Ⅱ型(herpes simplex virus type 2, HSV2)复制周期中的作用,并在此基础上以ERK通路关键激酶MEK蛋白为切入点,解析MEK1和MEK2亚型在由HSV2介导的ERK通路活化过程中不同激活时相中的作用,以期深入了解MEK激酶不同亚型的功能,为揭示ERK信号通路参与调控HSV2复制的作用机制提供重要的实验依据。 方法： 1.通过观察病毒感染宿主细胞引起的细胞病变效(cytopathetic effect, CPE),建立HSV2感染HEK293细胞(human embryonic kidney 293 cells)的病毒—细胞模型； 2.用MOI=1.5的HSV2感染HEK293细胞,收集感染后Omin、5min、10min、20min、30min以及1h、2h、4h、8h、12h、24h、48h时间点的细胞及上清液,用Western blot方法检测细胞中ERK蛋白的磷酸化水平,即p-ERK1/2(phospho-ERK1/2,p-ERK1/2)水平,用终点滴定法检测上清液中病毒感染滴度(50% tissue culture infectious dose,TCID50)观察HSV2复制对ERK通路活化的影响； 3.用MEK1/2特异性抑制剂U0126预处理HEK293细胞1h,再用MOI=1.5的HSV2感染细胞,分别收集与方法2相同的感染时间点的细胞及上清液,检测各时间点p-ERK1/2水平及上清液中病毒TCID50,观察U0126对ERK通路活化的影响及阻断ERK通路对HSV2复制的影响； 4.在距离紫外灯管17cm处用紫外线照射(Ultraviolet-irradiated, UV) HSV230min将其灭活,即UV-HSV2(以下也称为死病毒)。然后用MOI=10的死、活病毒分别感染HEK293细胞,收集感染后20min和4h的细胞,检测各时间点p-ERK1/2水平,以观察死、活病毒对ERK通路不同激活时相的影响； 5.分别设计并合成针对人类mek1、mek2基因的特异性的小干扰RNA (smallinterfering RNA, siRNA)及其突变型MKE1 siRNA (mutant MKE1 siRNA, siMut1)、MKE2 siRNA (mutant MKE2 siRNA,siMut2),用INTERFERin转染试剂盒将其分别转染HEK293细胞48h后,再分别用MOI=10的死、活病毒感染细胞,收集感染后20min、4h的细胞并用Western blot方法检测p-ERK1/2水平,观察分别敲降(knock down) MEK1、MEK2后对病毒感染引起的ERK通路早期时相和晚期时相激活的影响。 结果： 1.HSV2能够感染HEK293细胞并引起典型的细胞病变效应,HEK293细胞可以作为易感细胞用于后续实验； 2.HSV2感染可引起HEK293细胞的ERK通路激活,且呈双相激活,p-ERK1/2水平分别在病毒感染后20min和411达到高峰,可选用这两个时间点分别代表ERK通路激活的早期和晚期时相； 3.U0126可以显著抑制HSV2感染引起的ERK通路中的p-ERK1/2水平的增高,且抑制ERK通路能够明显抑制细胞上清液中的病毒滴度水平； 4.HSV2活病毒感染能引起ERK通路早期时相和晚期时相激活,而死病毒仅能引起ERK通路早期时相激活； 5.单独敲降细胞MEK1对活病毒引起的ERK通路的早期时相和晚期时相激活均有显著抑制作用,且对死病毒引起的早期时相的激活有抑制作用。而单独敲降细胞MEK2对活病毒引起的早期时相和晚期时相激活及死病毒引发的晚期时相激活均未见显著影响。 结论： 1.HSV2的复制依赖细胞ERK通路的激活； 2.HSV2感染宿主细胞引起ERK通路双相激活的机制是不同的,ERK通路的早期时相激活可能与病毒的吸附、穿入和脱壳过程相关；而晚期时相激活可能与病毒在宿主细胞中的生物合成过程相关。因此,细胞ERK通路的不同激活时相可能是其调控HSV2复制一种重要机制； 3.MEK1激酶在HSV2介导的ERK通路双相激活中发挥了关键的正调控作用,其调控机制可能是通过MEK1-ERKs级联激活方式来调控的HSV2介导的ERK通路双相激活。
[Abstract]:Purpose: In this study, the role of Raf/ MEK/ ERK (ERK) in the replication cycle of herpes simplex virus type 2 (HSV2) was clarified, and the key kinase MEK protein of ERK was cut in. The role of the MEK1 and MEK2 subtypes in the activation of the ERK pathway mediated by HSV2 in the activation of the ERK pathway mediated by the HSV2 is analyzed, with a view to further understanding the function of the different subtypes of the MEK kinase, and to provide an important experimental basis for revealing the mechanism of the ERK signaling pathway to participate in the regulation and regulation of the replication of the HSV2. It was reported. Methods:1. The virus of HEK293 cells (HEK293 cells) infected with HSV2 was established by observing the cytopathogenic effect (CPE) caused by the host cell of the virus. 2. The HEK293 cells were infected with HSV2 with MOI = 1.5, and after infection, Omin, 5min, 10min, 20min and 30min were collected. The level of ERK protein was detected by Western blot, that is, p-ERK1/2 (phospho-ERK1/2, p-ERK1/2). The HSV2 replication pair ERK is observed by the DSE (TCID50) Effect of pathway activation;3. The HEK293 cells were pre-treated with MEK1/2-specific inhibitor U0126 for 1 h, and the cells were infected with HSV2 with MOI = 1.5, and the cells and the supernatant at the same time of infection as method 2 were collected, and the p-ERK1/2 level and the supernatant at each time point were detected. The effect of U0126 on the activation of ERK pathway and the blocking of ERK via TCID50 The effect of HSV2 replication;4. Inactivation by UV-irradiation (UV) HSV230min at a distance of 17 cm from the UV lamp, i.e., UV-HSV 2 (also referred to as dead virus). Then, with MOI = 10, the live virus was infected with HEK293 cells respectively, and the cells of 20 min and 4 h after infection were collected, and p-ERK1/2 levels were detected for each time point to observe the death and live virus to ERK. The effect of the different activation phases of the path;5. The specific small interfering RNA (siRNA) and its mutant MKE1 siRNA (siMu1) and MKE2 siRNA (mutant MKE1 siRNA, siMu2) for human mek1 and mek2 genes were designed and synthesized. Cells were collected for 20 min and 4 h after infection, and the level of p-ERK1/2 was detected by Western blot. phase-time phase Results:1. HSV2 can infect HEK293 cells and cause typical cytopathic effect, HEK29 3 cells can be used as susceptible cells for subsequent experiments;2. The HSV2 infection can cause the ERK passage of the HEK293 cells to be activated and the two-phase activation, p-ERK1/2 levels, respectively, The two time points can be selected when the peak is reached at 20 min and 411 after the virus infection 3. U0126 can significantly inhibit the level of p-ERK1/2 in ERK pathway caused by HSV2 infection and inhibit ERK. the pathway can significantly inhibit the level of virus titer in the supernatant of the cell;4. the HSV2 live virus infection can cause the early phase and the late phase of the ERK pathway Phase activation, whereas the dead virus can only cause early phase activation of the ERK pathway;5. The individual knock-down cell MEK1 is responsible for the live virus The early phase and late phase activation of ERK pathway were significant. Inhibition, and inhibition of the activation of the early phase caused by the dead virus, while the individual knock-down cell MEK2 has an early phase and a late phase of the live virus. phase-time phase There was no significant effect on the activation and late phase activation of the activation and death of the virus Conclusion:1. The replication of HSV2 depends on the activation of ERK pathway. And the early phase activation of the ERK pathway may be associated with the adsorption, penetration, and dehulling of the virus. Range-related; while late-phase activation may be related to the process of the biosynthesis of the virus in the host cell. The different activation phases of the ERK pathway may be an important mechanism for regulating the replication of HSV2; K1 kinase plays a key role in the two-phase activation of the ERK pathway mediated by HSV2, and its regulatory mechanism may be through ME
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R373
本文编号：2434571
[Abstract]:Purpose: In this study, the role of Raf/ MEK/ ERK (ERK) in the replication cycle of herpes simplex virus type 2 (HSV2) was clarified, and the key kinase MEK protein of ERK was cut in. The role of the MEK1 and MEK2 subtypes in the activation of the ERK pathway mediated by HSV2 in the activation of the ERK pathway mediated by the HSV2 is analyzed, with a view to further understanding the function of the different subtypes of the MEK kinase, and to provide an important experimental basis for revealing the mechanism of the ERK signaling pathway to participate in the regulation and regulation of the replication of the HSV2. It was reported. Methods:1. The virus of HEK293 cells (HEK293 cells) infected with HSV2 was established by observing the cytopathogenic effect (CPE) caused by the host cell of the virus. 2. The HEK293 cells were infected with HSV2 with MOI = 1.5, and after infection, Omin, 5min, 10min, 20min and 30min were collected. The level of ERK protein was detected by Western blot, that is, p-ERK1/2 (phospho-ERK1/2, p-ERK1/2). The HSV2 replication pair ERK is observed by the DSE (TCID50) Effect of pathway activation;3. The HEK293 cells were pre-treated with MEK1/2-specific inhibitor U0126 for 1 h, and the cells were infected with HSV2 with MOI = 1.5, and the cells and the supernatant at the same time of infection as method 2 were collected, and the p-ERK1/2 level and the supernatant at each time point were detected. The effect of U0126 on the activation of ERK pathway and the blocking of ERK via TCID50 The effect of HSV2 replication;4. Inactivation by UV-irradiation (UV) HSV230min at a distance of 17 cm from the UV lamp, i.e., UV-HSV 2 (also referred to as dead virus). Then, with MOI = 10, the live virus was infected with HEK293 cells respectively, and the cells of 20 min and 4 h after infection were collected, and p-ERK1/2 levels were detected for each time point to observe the death and live virus to ERK. The effect of the different activation phases of the path;5. The specific small interfering RNA (siRNA) and its mutant MKE1 siRNA (siMu1) and MKE2 siRNA (mutant MKE1 siRNA, siMu2) for human mek1 and mek2 genes were designed and synthesized. Cells were collected for 20 min and 4 h after infection, and the level of p-ERK1/2 was detected by Western blot. phase-time phase Results:1. HSV2 can infect HEK293 cells and cause typical cytopathic effect, HEK29 3 cells can be used as susceptible cells for subsequent experiments;2. The HSV2 infection can cause the ERK passage of the HEK293 cells to be activated and the two-phase activation, p-ERK1/2 levels, respectively, The two time points can be selected when the peak is reached at 20 min and 411 after the virus infection 3. U0126 can significantly inhibit the level of p-ERK1/2 in ERK pathway caused by HSV2 infection and inhibit ERK. the pathway can significantly inhibit the level of virus titer in the supernatant of the cell;4. the HSV2 live virus infection can cause the early phase and the late phase of the ERK pathway Phase activation, whereas the dead virus can only cause early phase activation of the ERK pathway;5. The individual knock-down cell MEK1 is responsible for the live virus The early phase and late phase activation of ERK pathway were significant. Inhibition, and inhibition of the activation of the early phase caused by the dead virus, while the individual knock-down cell MEK2 has an early phase and a late phase of the live virus. phase-time phase There was no significant effect on the activation and late phase activation of the activation and death of the virus Conclusion:1. The replication of HSV2 depends on the activation of ERK pathway. And the early phase activation of the ERK pathway may be associated with the adsorption, penetration, and dehulling of the virus. Range-related; while late-phase activation may be related to the process of the biosynthesis of the virus in the host cell. The different activation phases of the ERK pathway may be an important mechanism for regulating the replication of HSV2; K1 kinase plays a key role in the two-phase activation of the ERK pathway mediated by HSV2, and its regulatory mechanism may be through ME
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R373
【参考文献】
中国期刊全文数据库 前2条
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2 王波;丁丽新;邓娟;张浩;朱萌;易婷;刘静;徐萍;鲁凤民;彭宜红;;MEK1/2抑制剂U0126抑制肠道病毒71型的复制[J];中国生物化学与分子生物学报;2010年06期
，本文编号：2434571
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