人巨细胞病毒感然对MRC-5细胞自噬的影响

发布时间：2018-04-05 02:26

本文选题：自噬　切入点：人巨细胞病毒　出处：《江苏大学》2017年硕士论文

【摘要】：目的:人巨细胞病毒(Human cytomegalovirus,HCMV)是疱疹病毒科(Herpesviridea)β亚科的一员,可在人群中广泛传播,在一些人群中血清阳性率高达90%。虽然健康人群感染HCMV通常是无症状的,但是它是免疫功能低下的人群中发病率和死亡率的重要原因,如艾滋病患者、骨髓及其他器官移植受者,它可导致危及生命的感染,感染可损害移植器官。此外,HCMV先天性感染是引起出生缺陷最主要的病毒,可引起多系统脏器功能的损伤。自噬在细胞的许多活动,如发育、分化、存活及细胞内稳态,起着至关重要的作用。除了对细胞内部和外部压力发生反应外,自噬还可以构成细胞免疫。它作为一种必不可少的宿主抵抗病毒感染的组成部分,是通过调节病原体的降解、先天性免疫及适应性免疫来实现的。病毒已经进化出多种方式来躲避自噬的攻击和操纵自噬的机制,从而使自噬有利于自身的复制。在本研究中,将重点观察人巨细胞病毒(Human cytomegalovirus,HCMV)在人胚肺成纤维细胞(MRC-5细胞)中对自噬的影响,以及探讨病毒HCMV是怎样调节自噬的。方法:1.检测在MRC-5细胞感染HCMV后的不同时间点的自噬水平的变化,及m TOR信号通路下游蛋白质的表达水平。将MOI=1的HCMV接种MRC-5细胞(人胚肺成纤维细胞),以未接种病毒的细胞为空白对照组,感染12h、24h、36h、48h、60h后分别做以下检测:1.1检测自噬水平的变化:利用流式细胞仪定量检测吖啶橙荧光颗粒阳性细胞数,并用Western Blot检测自噬相关蛋白;1.2检测m TOR信号通路下游分子的表达水平:运用Western Blot检测m TOR信号通路下游关键分子磷酸化的p70S6K(即p70S6K-p)及磷酸化的4E-BP1(即4E-BP1-p)的表达水平。2.为了进一步研究宿主细胞中的自噬对病毒复制的影响,可运用荧光定量PCR检测HCMV DNA的拷贝数。结果:HCMV感染的MRC-5细胞LC3、Beclin1蛋白表达水平先升高再下降,与流式细胞仪检测结果一致;同时,HCMV感染的MRC-5细胞4E-BP-1-p、p70S6K-p蛋白的表达水平先降低后升高;HCMV DNA拷贝数是先增加后降低。结论:HCMV感染可先促进后抑制MRC-5细胞自噬,其机制可能与m TOR有关,且自噬在一定程度上有利于HCMV的复制。
[Abstract]:Objective: human cytomegalovirus human cytomegalovirus (HCMV) is a member of Herpesviridae 尾 subfamily of Herpesviridae, which can be widely spread in the population. In some people, the positive rate of serum is as high as 90%.Although HCMV infection in healthy people is usually asymptomatic, it is an important cause of morbidity and mortality in people with low immune function, such as AIDS patients, bone marrow and other organ transplant recipients, which can lead to life-threatening infections.Infection can damage transplanted organs.In addition, congenital infection of HCMV is the most important virus causing birth defects, which can cause multiple system organ function damage.Autophagy plays a crucial role in many cellular activities, such as development, differentiation, survival and homeostasis.In addition to responding to internal and external pressures, autophagy can also form cellular immunity.As an essential part of host resistance to virus infection, it is achieved by regulating pathogen degradation, innate immunity and adaptive immunity.Viruses have evolved multiple ways to avoid autophagy attacks and manipulate the mechanism of autophagy so that autophagy is conducive to self-replication.In this study, the effect of human cytomegalovirus (HCMV) on autophagy in human embryonic lung fibroblasts (MRC-5) and how the virus HCMV regulates autophagy was studied.Method 1: 1.The level of autophagy and the expression of protein downstream of m TOR signaling pathway were detected at different time points after HCMV infection in MRC-5 cells.The HCMV of MOI=1 was inoculated with MRC-5 cells (human embryonic lung fibroblasts).The changes of autophagy were detected as follows: flow cytometry was used to quantitatively detect the number of acridine orange fluorescent granulosa positive cells after infection for 12 h, 24 h, 36 h, 48 h and 60 h, respectively.The expression level of downstream molecules in m TOR signaling pathway was detected by Western Blot. The expression levels of phosphorylated p70S6K (p70S6K-p) and phosphorylated 4E-BP1-p1 (4E-BP1-p) were detected by Western Blot.In order to further study the effect of autophagy on viral replication in host cells, the copy number of HCMV DNA can be detected by fluorescence quantitative PCR.Results the expression level of LC3 / Beclin1 protein in MRC-5 cells infected with 1% HCMV was increased first and then decreased, which was consistent with the results of flow cytometry, while the expression level of p70S6K-p protein in 4E-BP-1-pnpnp70S6K-p cells decreased first and then increased then decreased.Conclusion the infection of MRC-5 can promote and then inhibit the autophagy of MRC-5 cells. The mechanism may be related to m TOR, and autophagy is beneficial to the replication of HCMV to some extent.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R373

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