人偏肺病毒入胞机制的研究

发布时间：2018-09-10 13:26

【摘要】：人偏肺病毒(human metapneumovirus,hMPV)是引起婴幼儿呼吸系统感染的重要病原体,主要引起婴幼儿上、下呼吸道感染。虽然目前研究认为副黏病毒科病毒通过与细胞膜表面受体结合启动入胞,但是hMPV的具体入胞途径并不是很清楚。病毒进入宿主细胞的方式主要有两种:膜融合和胞吞。本课题组首先对hMPV是以膜融合还是胞吞入胞进行了研究。通过间接免疫荧光、R18融合实验、R18/DiOC双波长成像实验证明了hMPV在进入Vero E6细胞时存在胞吞的入胞方式。胞吞需要经历一系列复杂的过程,而脂筏(lipid raft)广泛的存在于细胞膜表面,因此本课题组对hMPV胞吞过程中是否有脂筏的参与进行了研究。通过对hMPV入胞过程的形态学研究、化学阻断剂处理等方面证明了脂筏参与了hMPV的入胞过程并在入胞过程中发挥着重要的作用。第一部分:人偏肺病毒以胞吞方式入胞的研究目的:探究hMPV在Vero E6细胞中的入胞方式,通过对其入胞机制的研究寻找出一种有效的阻断hMPV感染的的手段。方法:首先将100μl h MPV(MOI=10)接种到Vero E6细胞,分别在感染后的0 min、30 min、60 min、120 min洗去游离的hMPV并固定细胞;用带绿色荧光的抗体标记病毒包膜的融合蛋白(F蛋白),用带红色荧光的抗体标记位于胞核的核心蛋白(n蛋白),通过激光共聚焦显微镜观察在感染后0min、30min、60min、120min时进入veroe6细胞的hmpv数量及其在细胞中所处的位置。然后用r18染料对hmpv包膜染色,用染色后的hmpv感染veroe6细胞并用激光共聚焦显微镜实时拍摄4h以观察hmpv在veroe6细胞胞质中是否有膜融合的发生。最后用dioc和r18两种染料对hmpv包膜染色,用染色后的hmpv感染veroe6细胞并用激光共聚焦显微镜实时拍摄2h以观察hmpv在veroe6细胞发生膜融合的位置。结果:荧光抗体对hmpvf蛋白和n蛋白进行标记后发现,在不同的时间点均能观察到在veroe6细胞中f蛋白和n蛋白存在共定位并且随着时间的增加共定位点数量也逐渐增加。此结果表明hmpv在进入veroe6细胞时可以保持病毒颗粒结构的完整。r18融合实验观察到细胞出现红色荧光并且随着时间的增加红色荧光逐渐增多,说明hmpv在入胞过程中发生膜融合。dioc/r18融合实验中整个过程均有红色荧光出现并逐渐向胞质内移动,而绿色荧光比红色荧光出现晚,黄色荧光在30min以后出现一个急剧上升的过程,说明在30min后hmpv包膜与细胞膜发生膜融合并且膜融合发生在细胞质中。结论:本实验证明了hmpv存在胞吞的方式进入veroe6细胞,在实验过程中,首先用间接免疫荧光证明了hmpv可以完整的进入细胞;然后用r18融合实验证明了hmpv在进入细胞时与细胞膜发生了膜融合;最后用r18/dioc融合实验证明了hmpv入胞30min时发生膜融合并且膜融合发生的部位在胞质内,这些实验均证实了hmpv存在胞吞的方式进入Vero E6细胞。第二部分:脂筏在人偏肺病毒感染宿主细胞中的作用机制研究目的:探究脂筏是否参与了人偏肺病毒入胞的过程,通过对脂筏的研究进一步了解h MPV入胞过程。方法:h MPV接种病毒后在培养基中加入霍乱毒素亚单位(CTB)同时作用6HBE细胞,分别在感染后的0 min、30 min、60 min、120 min固定细胞,用带红色荧光的抗体标记h MPV融合蛋白(F蛋白),激光共聚焦显微镜观察CTB与h MPV在不同时间点的位置关系;分别用1mg/ml、5 mg/ml、7.5 mg/ml MβCD和10μg/ml,20μg/ml,30μg/ml Nystatin处理16HBE细胞后感染h MPV,感染24 h后用荧光定量PCR检测不同处理组h MPV滴度。将50μg/ml、100μg/ml外源性胆固醇加入5 mg/ml MβCD处理后的16HBE细胞,接毒后24 h用荧光定量PCR检测h MPV滴度。结果:h MPV在进入宿主细胞时与脂筏标记物霍乱毒素亚单位存在共定位,并且随着时间的增加,共定位点逐渐增加;h MPV伴随着CTB逐渐向胞核方向移。MβCD处理后,随着药物浓度的逐渐升高h MPV滴度分别降低了81%、90%和98%;Nystatin处理后,随着药物浓度的逐渐升高h MPV滴度分别降低了44%、63%和89%。向5 mg/ml MβCD处理后的16HBE细胞中加入外源性胆固醇,当胆固醇浓度达到100μg/ml时,h MPV感染显著升高;添加外源性胆固醇后h MPV滴度时药物处理组的4倍。结论:本实验证明了脂筏参与了人偏肺病毒胞吞过程并在胞吞过程中发挥重要作用。首先用CTB来标记脂筏,通过CTB来观察不同时间点脂筏与h MPV位置关系;然后用了MβCD、Nystatin两种破坏脂筏的药物处理细胞来检测脂筏破坏对h MPV感染的影响;最后通过外源性胆固醇补救检测添加外源性胆固醇是否可以恢复h MPV感染,这些实验均证实了脂筏参与了人偏肺病毒胞吞过程。
[Abstract]:Human metapneumovirus (hMPV) is an important pathogen causing respiratory tract infections in infants and young children. It mainly causes upper and lower respiratory tract infections in infants and young children. There are two main types of host cells: membrane fusion and endocytosis. In this study, we first studied whether hMPV is membrane fusion or endocytosis. Indirect immunofluorescence, R18 fusion, and R18/DiOC dual-wavelength imaging demonstrated that hMPV has endocytosis when it enters Vero E6 cells. Lipid raft is widely distributed on the surface of cell membrane, so we have studied whether lipid raft is involved in the process of hMPV endocytosis. Part I: The purpose of this study is to explore the entry mode of hMPV into Vero E6 cells by endocytosis, and to find an effective way to block the infection of hMPV through the study of its entry mechanism. Free hMPV was washed and fixed at 60 min, 120 min. The fusion protein (F protein) coated with green fluorescent antibody was labeled and the core protein (n protein) located in the nucleus was labeled with red fluorescent antibody. The number of hMPV entering veroe6 cells at 0 min, 30 min, 60 min and 120 min after infection and the number of hMPV entering veroe6 cells were observed by laser confocal microscopy. The location of hMPV in the cells was observed by R18 dye. the cells were infected with the stained hMPV for 4 hours. the fusion of hMPV in the cytoplasm of veroe 6 cells was observed by confocal laser microscopy. finally, the hMPV envelope was stained with dioc and R18 dyes, and veroe 6 cells were infected with the stained hMPV Results: Fluorescent antibody labeling of hMPV F protein and N protein showed that co-localization of F protein and N protein was observed in veroe 6 cells at different time points and the number of co-localization points increased with time. The results showed that Hpv maintained the integrity of virus granule structure when it entered veroe 6 cells. In R18 fusion experiment, red fluorescence was observed and increased gradually with the increase of time, indicating that membrane fusion occurred during the process of entry. In dioc / R18 fusion experiment, red fluorescence was observed throughout the whole process. The green fluorescence appeared later than the red fluorescence, and the yellow fluorescence showed a sharp rise after 30 minutes, indicating that the membrane fusion between the hMPV envelope and the cell membrane occurred in the cytoplasm after 30 minutes. In the process of experiment, indirect immunofluorescence was used to prove that hMPV could enter the cell completely; then R18 fusion experiment was used to prove that hMPV fused with the cell membrane when it entered the cell; finally, R18 / dioc fusion experiment was used to prove that the membrane fusion took place in the cytoplasm when hMPV entered the cell for 30 minutes, and the location of membrane fusion took place in the cytoplasm. Objective: To investigate the role of lipid rafts in the process of human metapneumovirus (HMPV) entry into Vero E6 cells, and to further understand the process of HMPV entry through the study of lipid rafts. Cholera toxin subunit (CTB) was added to 6 HBE cells at the same time. Fixed cells were immobilized at 0, 30, 60 and 120 minutes after infection, labeled with red fluorescent antibodies for fusion protein of h-MPV (F protein), and the relationship between CTB and h-MPV at different time points was observed by confocal laser microscopy. H MPV titer was detected by fluorescence quantitative PCR 24 hours after treatment with 20 ug/ml, 30 ug/ml Nystatin and 15 mg/ml exogenous cholesterol and 5 mg/ml M beta CD respectively. The co-localization of the labeled cholera toxin subunits was found, and the co-localization point increased gradually with the increase of time; the H MPV gradually moved toward the nucleus with the increase of CTB. The titer of H MPV decreased by 81%, 90% and 98% respectively with the increase of drug concentration after Mbeta CD treatment; the titer of H MPV decreased by 90% and 98% respectively with the increase of drug concentration after Nystatin treatment. When the concentration of cholesterol reached 100 ug/ml, the infection of H MPV was significantly increased, and the titer of H MPV was 4 times as high as that of the drug treatment group. First, CTB was used to label lipid rafts, and CTB was used to observe the location relationship between lipid rafts and H MPV at different time points. Then, the effect of lipid raft destruction on the infection of H MPV was detected by using two drug-treated cells, M-beta-CD and Nystatin. Finally, the possibility of adding exogenous cholesterol was detected by exogenous cholesterol salvage test. In order to restore h MPV infection, these experiments confirmed that lipid rafts were involved in the process of human PPV endocytosis.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R373

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