水痘—带状疱疹病毒诱导人神经母细胞瘤细胞的自噬作用观察
发布时间：2018-08-22 07:57
【摘要】：背景水痘—带状疱疹病毒(varicella-zoster virus,VZV)属α疱疹病毒亚科,首次感染人体后能引起水痘或呈隐性感染状态,在对人体的初次感染后,病毒沿感觉神经侵入脊神经节或脑神经感觉神经节内并潜伏于其中。当机体免疫力低下时病毒被再激活而引起带状疱疹。VZV具有亲神经性,可引起带状疱疹后遗神经痛(postherpetic neuralgia,PHN),PHN在临床上十分棘手,给患者的心理和生理带来严重影响。自噬是一种高度保守的细胞内降解途径,其依赖溶酶体途径降解细胞内受损的细胞器和错误折叠的蛋白质以维持内环境稳态。自噬也参与到固有免疫和先天性免疫清除入侵病原体的应答过程中。近年来研究表明细胞可以激活自噬来控制人类疱疹病毒感染。然而人类疱疹病毒进化出多种机制来逃避、抑制、破坏甚至利用细胞自噬。本文观察了 VZV在人神经母细胞瘤细胞株(SHSY5Y)中培养及增殖的特点和VZV感染SHSY5Y细胞后的生物学效应,探讨感染后自噬发生的规律,为有效地干预和治疗VZV感染提供新思路。目的本研究的目的是探讨VZV在SHSY5Y细胞中培养及增殖的特点,观察VZV感染SHSY5Y细胞后的生物学效应,探讨感染后自噬发生的规律和特点,为有效地干预和治疗VZV感染提供新思路。方法1.于普通倒置显微镜和电子显微镜下观察被感染细胞的形态变化,利用CCK-8法检测VZV感染对细胞增殖的影响;比较不同感染复数(MOI)和不同浓度胎牛血清(FBS)维持液对病毒增殖的影响;2.利用单丹(磺)酰戊二胺(MDC)染色法检测自噬体的形成,通过流式细胞术测定VZV感染细胞后不同时间点(24h、48h、72h、96h)自噬相关蛋白微管结合蛋白-1 轻链-3B(microtubule-associated protein 1 light chain 3B,MAP1LC3B,又称为LC3B)和SQSTM1/p62的表达情况;3.提取VZV病毒中的DNA,利用荧光定量PCR检测VZV感染细胞后不同时间点(24h、48h、72h、96h)的病毒DNA拷贝数。结果1.普通倒置显微镜下观察细胞病变效应(cytopathic effect,CPE,从24至96h,细胞病变数量增加和病变程度加重。电镜观察显示:感染48h后核染色质固缩、浓染,线粒体肥大及增生,胞质空泡化和自噬小体形成,可见包装成熟的病毒颗粒。CCK-8法提示VZV感染24h后细胞增殖被抑制,各实验组存活率与对照组比较均有统计学差异(P0.05)。在2%胎牛血清的维持液中用MOI为1的VZV感染SHSY5Y细胞,能收获较高滴度的病毒。2.MDC染色法显示VZV感染细胞后自噬体数量增加,流式细胞术检测结果表明VZV感染组的自噬相关蛋白LC3B-Ⅱ蛋白的表达增加,而p62蛋白的表达出现下降,与正常对照组比较均有统计学差异(P0.05)。3.荧光定量PCR结果表明VZV感染SHSY5Y细胞后72h的DNA拷贝数明显高于其余三组(24h、48h、96h)(P0.05)。结论1.本实验初步建立了 VZV感染SHSY5Y细胞的体外模型,并利用该模型观察病毒感染对细胞生物性状的影响,为后续探讨VZV的致病机制奠定了基础。2.VZV感染SHSY5Y细胞后有诱导自噬的作用,可在细胞中观察到自噬小体和自噬相关蛋白的增加,而且自噬流过程通畅,说明VZV感染SHSY5Y细胞后自噬活性增强。此外,SHSY5Y细胞自噬活性的增强也有可能促进VZV的复制,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.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R752.12
本文编号：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.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R752.12
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