新型呼肠病毒BYD1株诱导细胞凋亡的分子机制及其与致病性关系的研究

发布时间：2018-05-03 16:31

本文选题：呼肠病毒 + 凋亡　；参考：《中国人民解放军军事医学科学院》2008年博士论文

【摘要】： 2003年7月本实验室从SARS患者标本中分离得到1株新型呼肠病毒BYD1株[1]。这是继1982年国内报道分离出呼肠病毒21年后再次分离得到呼肠病毒[2]。与人类疾病相关的呼肠病毒以往曾经在婴儿和儿童病患体内分离得到,患者出现了致死性脑炎、致死性肺炎、间歇性发热、腹泻等症状[3,4,5,6]。尽管如此,一般认为该病毒对成人不致病,仅能造成隐性感染,没有明显临床症状。长期以来国内对该病毒研究甚少,已有的临床病例和基础研究的报道基本来自国外。自SARS患者标本中分离出BYD1株病毒后,已引起国内对该病毒的注意[7],同时本实验室对BYD1株病毒也开展了一系列研究[8,9,10]。呼肠病毒与致病性相关的重要特征是某些毒株能够在体外诱导多种细胞系凋亡[11,12,13]。本论文将对BYD1株病毒诱导细胞凋亡的能力、诱导凋亡的分子机制、病毒致病性及凋亡和致病性之间的关系进行研究。本研究用105TCID50的BYD1株病毒感染HeLa细胞,12h后用胰酶消化细胞致单个,采用碘化丙啶(PI)和异硫氰酸荧光素(FITC)标记的Ca2+依赖性磷脂结合蛋白(Annexin-V)对感染细胞进行染色,然后经流式细胞仪检测细胞凋亡率。通过成组资料的t检验分析,BYD1株病毒感染的HeLa细胞凋亡率为14.32±0.71,模拟感染细胞凋亡率为4.06±0.23,t值为26.69,大于t0.05。分析表明BYD1株病毒具有诱导细胞凋亡能力。呼肠病毒在宿主细胞内复制一代所需时间不超过16小时[14],但本研究发现BYD1株病毒颗粒对凋亡的诱导早在感染后12小时就可以检测到,因此推测病毒对细胞凋亡的诱导发生在病毒复制完成之前。凋亡的发生可能不是由于病毒颗粒的复制,而是与病毒的某些蛋白分子有关。有研究指出,呼肠病毒结构蛋白σ1和μ1具有病毒诱导细胞凋亡的能力。μ1、σ3和σ1是呼肠病毒重要的外衣壳蛋白成份,它们参与病毒入侵宿主细胞的过程:呼肠病毒通过类似受体介导的内吞方式入侵到细胞浆后,σ3蛋白被降解。感染性亚病毒颗粒(infectious subvirion particles,ISVPs)形成,σ1蛋白构象随之发生改变,μ1蛋白也被裂解为与病毒颗粒相连的δ和φ片段[15,16,17]。然后病毒释放出完整的病毒核心,在胞浆中开始子代病毒复制。为研究BYD1株病毒引起细胞凋亡的分子机制,本研究选取σ1全长基因、μ1全长基因、以及δ和φ基因片段,将其克隆到真核表达载体pEGFP-C3中,构建成重组质粒pEGFP-C-σ1(1-461)、pEGFP-C-μ1(1-708)、pEGFP-C-μ1(1-675)和pEGFP-C-μ1(582-708)。然后将每一个重组质粒分别转染293T细胞。于转染后18h观察细胞病变状态;与转染后18h收获细胞用于细胞凋亡率的检测;于转染后48h收获细胞,用于电镜下的形态学分析。转染后18h,在普通光镜下可见宿主细胞出现明显病变,细胞边界不清楚、细胞皱缩、甚至破碎。同一病变细胞在荧光显微镜下可见有融合蛋白的表达。转染后48h,电镜形态学观察发现细胞出现了典型凋亡特征,可观察到处于凋亡早期、中期和晚期的细胞。用流式细胞仪检测转染18h的细胞凋亡率,并进行统计分析,发现BYD1株病毒σ1蛋白、μ1蛋白、以及μ1(1-675)和φ片段都具有诱导细胞凋亡的能力。实验结果显示,σ1蛋白、μ1蛋白、以及μ1(1-675)和φ片段是BYD1株病毒诱导细胞凋亡的重要决定因子,这些蛋白单独或相互作用来起始凋亡信号。为研究新型呼肠病毒BYD1株的致病性与细胞凋亡的关系,本研究通过颅腔注射方式使2日龄乳鼠感染BYD1株病毒。每只乳鼠注射病毒原液20μl,逐日观察乳鼠发病情况。表征观察,乳鼠病程经历了急性发病期、缓和期以及症状二次加重的变化过程。在注射病毒后第8天处死动物,取出脏器,制成石蜡包埋组织切片,对组织切片进行组织病理分析和TUNEL法原位凋亡检测。结果显示,BYD1株病毒感染可对乳鼠中枢神经、心脏和肺等全身多脏器产生损害,新型呼肠病毒BYD1株的感染与动物中枢神经损伤、心肌炎和肺炎的发病关系密切;TUNEL法原位细胞凋亡检测,感染乳鼠海马组织可见大量凋亡细胞,说明BYD1株病毒诱导的细胞凋亡是造成组织损伤的一个直接原因。
[Abstract]:A novel reovirus BYD1 strain was isolated from SARS patients in July 2003 . It was reported in 1982 that the reovirus was isolated again after 21 years of isolation of reovirus . In the past , the virus has been isolated from SARS patients . It has been reported that the virus can only cause recessive infection and has no obvious clinical symptoms .

In this paper , we will study the relationship between the ability of BYD1 strain to induce apoptosis , the molecular mechanism of inducing apoptosis , the pathogenicity of the virus and the relationship between apoptosis and pathogenicity .

The apoptosis rate of HeLa cells infected by BYD1 strain was 14.32 卤 0.71 , the apoptosis rate of HeLa cells infected by BYD1 was 14.32 卤 0.71 , the apoptosis rate of infected cells was 4.06 卤 0.23 , t value was 26.69 , which was more than t0 . 05 . It was indicated that BYD1 strain had the ability to induce apoptosis .

It is suggested that the induction of apoptosis by BYD1 strain is not due to replication of virus particles , but it is related to certain protein molecules of the virus .

In order to study the molecular mechanism of cell apoptosis induced by BYD1 strain , we selected 蟽1 full - length gene , 渭1 full - length gene , and 未 and 蠁 gene fragments , and then cloned into eukaryotic expression vector ( 1 - 461 ) . After transfection , the cell apoptosis rate was observed . After transfection , the cell apoptosis rate was observed under microscope . The results showed that 蟽1 protein , 渭1 protein , and 渭 1 ( 1 - 675 ) and 蠁 fragment were the important determinants of apoptosis .

In order to study the relationship between the pathogenicity of BYD1 strain and apoptosis of the novel reovirus BYD1 strain , this study was conducted to infect BYD1 strain in 2 - day - old mice by injection of cranial cavity . Each mouse injected 20 渭l of virus stock solution to observe the incidence of neonatal mice .

The results showed that the infection of BYD1 strain could damage the central nervous system , heart and lung of the mice . The results showed that the infection of BYD1 strain could damage the central nervous system , heart and lung of the mice . The infection of BYD1 strain was closely related to the pathogenesis of central nerve injury , myocarditis and pneumonia .

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R373

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