本文選題：自噬　切入點(diǎn)：人巨細(xì)胞病毒　出處：《江蘇大學(xué)》2017年碩士論文

【摘要】：目的:人巨細(xì)胞病毒(Human cytomegalovirus,HCMV)是皰疹病毒科(Herpesviridea)β亞科的一員,可在人群中廣泛傳播,在一些人群中血清陽性率高達(dá)90%。雖然健康人群感染HCMV通常是無癥狀的,但是它是免疫功能低下的人群中發(fā)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,它可導(dǎo)致危及生命的感染,感染可損害移植器官。此外,HCMV先天性感染是引起出生缺陷最主要的病毒,可引起多系統(tǒng)臟器功能的損傷。自噬在細(xì)胞的許多活動(dòng),如發(fā)育、分化、存活及細(xì)胞內(nèi)穩(wěn)態(tài),起著至關(guān)重要的作用。除了對(duì)細(xì)胞內(nèi)部和外部壓力發(fā)生反應(yīng)外,自噬還可以構(gòu)成細(xì)胞免疫。它作為一種必不可少的宿主抵抗病毒感染的組成部分,是通過調(diào)節(jié)病原體的降解、先天性免疫及適應(yīng)性免疫來實(shí)現(xiàn)的。病毒已經(jīng)進(jìn)化出多種方式來躲避自噬的攻擊和操縱自噬的機(jī)制,從而使自噬有利于自身的復(fù)制。在本研究中,將重點(diǎn)觀察人巨細(xì)胞病毒(Human cytomegalovirus,HCMV)在人胚肺成纖維細(xì)胞(MRC-5細(xì)胞)中對(duì)自噬的影響,以及探討病毒HCMV是怎樣調(diào)節(jié)自噬的。方法:1.檢測(cè)在MRC-5細(xì)胞感染HCMV后的不同時(shí)間點(diǎn)的自噬水平的變化,及m TOR信號(hào)通路下游蛋白質(zhì)的表達(dá)水平。將MOI=1的HCMV接種MRC-5細(xì)胞(人胚肺成纖維細(xì)胞),以未接種病毒的細(xì)胞為空白對(duì)照組,感染12h、24h、36h、48h、60h后分別做以下檢測(cè):1.1檢測(cè)自噬水平的變化:利用流式細(xì)胞儀定量檢測(cè)吖啶橙熒光顆粒陽性細(xì)胞數(shù),并用Western Blot檢測(cè)自噬相關(guān)蛋白;1.2檢測(cè)m TOR信號(hào)通路下游分子的表達(dá)水平:運(yùn)用Western Blot檢測(cè)m TOR信號(hào)通路下游關(guān)鍵分子磷酸化的p70S6K(即p70S6K-p)及磷酸化的4E-BP1(即4E-BP1-p)的表達(dá)水平。2.為了進(jìn)一步研究宿主細(xì)胞中的自噬對(duì)病毒復(fù)制的影響,可運(yùn)用熒光定量PCR檢測(cè)HCMV DNA的拷貝數(shù)。結(jié)果:HCMV感染的MRC-5細(xì)胞LC3、Beclin1蛋白表達(dá)水平先升高再下降,與流式細(xì)胞儀檢測(cè)結(jié)果一致;同時(shí),HCMV感染的MRC-5細(xì)胞4E-BP-1-p、p70S6K-p蛋白的表達(dá)水平先降低后升高;HCMV DNA拷貝數(shù)是先增加后降低。結(jié)論:HCMV感染可先促進(jìn)后抑制MRC-5細(xì)胞自噬,其機(jī)制可能與m TOR有關(guān),且自噬在一定程度上有利于HCMV的復(fù)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.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R373

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 ;Hsp90 regulates processing of NF-κB2 p100 involving protection of NF-κB-inducing kinase (NIK) from autophagy-mediated degradation[J];Cell Research;2007年06期

2 ;Hsp90 inhibition results in autophagy-mediated proteasome-independent degradation of IκB kinase(IKK)[J];Cell Research;2006年11期

，

本文編號(hào)：1712792