水痘—帶狀皰疹病毒誘導(dǎo)人神經(jīng)母細(xì)胞瘤細(xì)胞的自噬作用觀察
發(fā)布時(shí)間：2018-08-22 07:57
【摘要】：背景水痘—帶狀皰疹病毒(varicella-zoster virus,VZV)屬α皰疹病毒亞科,首次感染人體后能引起水痘或呈隱性感染狀態(tài),在對(duì)人體的初次感染后,病毒沿感覺神經(jīng)侵入脊神經(jīng)節(jié)或腦神經(jīng)感覺神經(jīng)節(jié)內(nèi)并潛伏于其中。當(dāng)機(jī)體免疫力低下時(shí)病毒被再激活而引起帶狀皰疹。VZV具有親神經(jīng)性,可引起帶狀皰疹后遺神經(jīng)痛(postherpetic neuralgia,PHN),PHN在臨床上十分棘手,給患者的心理和生理帶來嚴(yán)重影響。自噬是一種高度保守的細(xì)胞內(nèi)降解途徑,其依賴溶酶體途徑降解細(xì)胞內(nèi)受損的細(xì)胞器和錯(cuò)誤折疊的蛋白質(zhì)以維持內(nèi)環(huán)境穩(wěn)態(tài)。自噬也參與到固有免疫和先天性免疫清除入侵病原體的應(yīng)答過程中。近年來研究表明細(xì)胞可以激活自噬來控制人類皰疹病毒感染。然而人類皰疹病毒進(jìn)化出多種機(jī)制來逃避、抑制、破壞甚至利用細(xì)胞自噬。本文觀察了 VZV在人神經(jīng)母細(xì)胞瘤細(xì)胞株(SHSY5Y)中培養(yǎng)及增殖的特點(diǎn)和VZV感染SHSY5Y細(xì)胞后的生物學(xué)效應(yīng),探討感染后自噬發(fā)生的規(guī)律,為有效地干預(yù)和治療VZV感染提供新思路。目的本研究的目的是探討VZV在SHSY5Y細(xì)胞中培養(yǎng)及增殖的特點(diǎn),觀察VZV感染SHSY5Y細(xì)胞后的生物學(xué)效應(yīng),探討感染后自噬發(fā)生的規(guī)律和特點(diǎn),為有效地干預(yù)和治療VZV感染提供新思路。方法1.于普通倒置顯微鏡和電子顯微鏡下觀察被感染細(xì)胞的形態(tài)變化,利用CCK-8法檢測(cè)VZV感染對(duì)細(xì)胞增殖的影響;比較不同感染復(fù)數(shù)(MOI)和不同濃度胎牛血清(FBS)維持液對(duì)病毒增殖的影響;2.利用單丹(磺)酰戊二胺(MDC)染色法檢測(cè)自噬體的形成,通過流式細(xì)胞術(shù)測(cè)定VZV感染細(xì)胞后不同時(shí)間點(diǎn)(24h、48h、72h、96h)自噬相關(guān)蛋白微管結(jié)合蛋白-1 輕鏈-3B(microtubule-associated protein 1 light chain 3B,MAP1LC3B,又稱為L(zhǎng)C3B)和SQSTM1/p62的表達(dá)情況;3.提取VZV病毒中的DNA,利用熒光定量PCR檢測(cè)VZV感染細(xì)胞后不同時(shí)間點(diǎn)(24h、48h、72h、96h)的病毒DNA拷貝數(shù)。結(jié)果1.普通倒置顯微鏡下觀察細(xì)胞病變效應(yīng)(cytopathic effect,CPE,從24至96h,細(xì)胞病變數(shù)量增加和病變程度加重。電鏡觀察顯示:感染48h后核染色質(zhì)固縮、濃染,線粒體肥大及增生,胞質(zhì)空泡化和自噬小體形成,可見包裝成熟的病毒顆粒。CCK-8法提示VZV感染24h后細(xì)胞增殖被抑制,各實(shí)驗(yàn)組存活率與對(duì)照組比較均有統(tǒng)計(jì)學(xué)差異(P0.05)。在2%胎牛血清的維持液中用MOI為1的VZV感染SHSY5Y細(xì)胞,能收獲較高滴度的病毒。2.MDC染色法顯示VZV感染細(xì)胞后自噬體數(shù)量增加,流式細(xì)胞術(shù)檢測(cè)結(jié)果表明VZV感染組的自噬相關(guān)蛋白LC3B-Ⅱ蛋白的表達(dá)增加,而p62蛋白的表達(dá)出現(xiàn)下降,與正常對(duì)照組比較均有統(tǒng)計(jì)學(xué)差異(P0.05)。3.熒光定量PCR結(jié)果表明VZV感染SHSY5Y細(xì)胞后72h的DNA拷貝數(shù)明顯高于其余三組(24h、48h、96h)(P0.05)。結(jié)論1.本實(shí)驗(yàn)初步建立了 VZV感染SHSY5Y細(xì)胞的體外模型,并利用該模型觀察病毒感染對(duì)細(xì)胞生物性狀的影響,為后續(xù)探討VZV的致病機(jī)制奠定了基礎(chǔ)。2.VZV感染SHSY5Y細(xì)胞后有誘導(dǎo)自噬的作用,可在細(xì)胞中觀察到自噬小體和自噬相關(guān)蛋白的增加,而且自噬流過程通暢,說明VZV感染SHSY5Y細(xì)胞后自噬活性增強(qiáng)。此外,SHSY5Y細(xì)胞自噬活性的增強(qiáng)也有可能促進(jìn)VZV的復(fù)制,提高病毒滴度。
[Abstract]:BACKGROUND Varicella-zoster virus (VZV) is a subfamily of alpha herpes virus. It can cause varicella or become a latent infection after the first infection. After the first infection, the virus invades the spinal ganglion or the sensory ganglion of the brain along the sensory nerve and lurks in it. VZV is neurophilic and can cause postherpetic neuralgia (PHN). PHN is very difficult in clinic and has serious psychological and physiological effects on patients. Autophagy is a highly conserved intracellular degradation pathway, which relies on lysosome pathway to degrade damaged cells. Autophagy is also involved in innate immunity and innate immune clearance of invading pathogens. Recent studies have shown that cells can activate autophagy to control human herpesvirus infection. However, human herpesvirus has evolved a variety of mechanisms to escape, suppress, destroy or even destroy. The characteristics of VZV culture and proliferation in human neuroblastoma cell line (SHSY5Y) and the biological effects of VZV infection on SHSY5Y cells were observed. The regularity of autophagy after infection was investigated. The aim of this study was to explore the culture of VZV in SHSY5Y cells. To observe the biological effects of VZV infection on SHSY5Y cells and to explore the regularity and characteristics of autophagy after infection, and to provide new ideas for effective intervention and treatment of VZV infection. Effects of different infection complex (MOI) and different concentrations of fetal bovine serum (FBS) maintenance fluid on virus proliferation were compared. 2. Autophagy was detected by MDC staining and autophagy-associated protein-1 was detected by flow cytometry at different time points (24h, 48h, 72h, 96h) after VZV infection. The expression of microtubule-associated protein 1 light chain 3B (MAP1LC3B) and SQSTM1/p62; 3. DNA was extracted from VZV virus and the copy number of virus DNA at different time points (24h, 48h, 72h, 96h) after VZV infection was detected by fluorescence quantitative PCR. Results 1. Cytopathy effect (cytopa) was observed under general inverted microscope. The number and degree of cytopathy increased from 24 to 96 hours after infection. Electron microscopic observation showed that nuclear chromatin pyknosis, dense staining, mitochondrial hypertrophy and hyperplasia, cytoplasmic vacuolation and autophagy corpuscles formation were seen after 48 hours of infection. CCK-8 method suggested that the proliferation of VZV cells was inhibited after 24 hours of infection, and the survival rate of each experimental group was inhibited. Compared with the control group, there was significant difference (P 0.05). SHSY5Y cells infected with VZV with MOI 1 in 2% fetal bovine serum could harvest higher titer of virus. 2. MDC staining showed that the number of autophages increased after VZV infection, and flow cytometry showed that the expression of autophagy-related protein LC3B-II increased in VZV infected group. The results of fluorescence quantitative PCR showed that the DNA copy number of SHSY5Y cells infected by VZV at 72 hours was significantly higher than that of the other three groups (24h, 48h, 96h) (P To investigate the effect of VZV infection on the biological characteristics of SHSY5Y cells, and to lay a foundation for further study on the pathogenesis of VZV infection. 2. VZV infection induced autophagy in SHSY5Y cells. The increase of autophagy bodies and autophagy-related proteins was observed in SHSY5Y cells, and the process of autophagy flow was smooth. The enhancement of autophagy activity of SY5Y cells may also promote the replication of VZV and increase the titer of virus.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R752.12
本文編號(hào)：2196487
[Abstract]:BACKGROUND Varicella-zoster virus (VZV) is a subfamily of alpha herpes virus. It can cause varicella or become a latent infection after the first infection. After the first infection, the virus invades the spinal ganglion or the sensory ganglion of the brain along the sensory nerve and lurks in it. VZV is neurophilic and can cause postherpetic neuralgia (PHN). PHN is very difficult in clinic and has serious psychological and physiological effects on patients. Autophagy is a highly conserved intracellular degradation pathway, which relies on lysosome pathway to degrade damaged cells. Autophagy is also involved in innate immunity and innate immune clearance of invading pathogens. Recent studies have shown that cells can activate autophagy to control human herpesvirus infection. However, human herpesvirus has evolved a variety of mechanisms to escape, suppress, destroy or even destroy. The characteristics of VZV culture and proliferation in human neuroblastoma cell line (SHSY5Y) and the biological effects of VZV infection on SHSY5Y cells were observed. The regularity of autophagy after infection was investigated. The aim of this study was to explore the culture of VZV in SHSY5Y cells. To observe the biological effects of VZV infection on SHSY5Y cells and to explore the regularity and characteristics of autophagy after infection, and to provide new ideas for effective intervention and treatment of VZV infection. Effects of different infection complex (MOI) and different concentrations of fetal bovine serum (FBS) maintenance fluid on virus proliferation were compared. 2. Autophagy was detected by MDC staining and autophagy-associated protein-1 was detected by flow cytometry at different time points (24h, 48h, 72h, 96h) after VZV infection. The expression of microtubule-associated protein 1 light chain 3B (MAP1LC3B) and SQSTM1/p62; 3. DNA was extracted from VZV virus and the copy number of virus DNA at different time points (24h, 48h, 72h, 96h) after VZV infection was detected by fluorescence quantitative PCR. Results 1. Cytopathy effect (cytopa) was observed under general inverted microscope. The number and degree of cytopathy increased from 24 to 96 hours after infection. Electron microscopic observation showed that nuclear chromatin pyknosis, dense staining, mitochondrial hypertrophy and hyperplasia, cytoplasmic vacuolation and autophagy corpuscles formation were seen after 48 hours of infection. CCK-8 method suggested that the proliferation of VZV cells was inhibited after 24 hours of infection, and the survival rate of each experimental group was inhibited. Compared with the control group, there was significant difference (P 0.05). SHSY5Y cells infected with VZV with MOI 1 in 2% fetal bovine serum could harvest higher titer of virus. 2. MDC staining showed that the number of autophages increased after VZV infection, and flow cytometry showed that the expression of autophagy-related protein LC3B-II increased in VZV infected group. The results of fluorescence quantitative PCR showed that the DNA copy number of SHSY5Y cells infected by VZV at 72 hours was significantly higher than that of the other three groups (24h, 48h, 96h) (P To investigate the effect of VZV infection on the biological characteristics of SHSY5Y cells, and to lay a foundation for further study on the pathogenesis of VZV infection. 2. VZV infection induced autophagy in SHSY5Y cells. The increase of autophagy bodies and autophagy-related proteins was observed in SHSY5Y cells, and the process of autophagy flow was smooth. The enhancement of autophagy activity of SY5Y cells may also promote the replication of VZV and increase the titer of virus.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R752.12
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